Serine protease inhibitors

ABSTRACT

The invention relates to a compound having the formula (I): R 1 SO 2 —B—X—Z—C(O)—Y, B is a bond, an amino acid of the formula —NR—CH[(CH 2 ) p C(O)OH]—C(O)— or an ester derivative thereof wherein p is 1, 2, or 3, Gly, D-1-Piq, D-3-Piq, D-1-Tiq, D-3-Tiq, D-Atc, Aic, or a L- or D-amino acid having a hydrophobic, basic or neutral side chain; X is an amino acid with a hydrophobic side chain, glutamine, serine, theronine, a cyclic amino acid optionally containing an additional heteroatom selected from N, O or S, and optionally substituted with (1-6C)alkyl, (1-6C)alkoxy, benzyloxy or oxo, or X is 2-amino-isobutyric acid, —NR 2 —CH 2 —C(O)— or the fragment (I) or (II), wherein n is 2, 3, or 4, W is CH or N and R 3  is H, (1-6C)alkyl or phenyl which groups may optionally be substituted with hydroxy, (1-6C)alkoxy, COOH, COO(1-6C)alkyl, CONH 2 , or halogen; Z is lysine or 4-aminocyclohexylglycine. The compounds of the invention have anticoagulant activity and can be used in treating or preventing thrombin-related diseases. The variable R 1  and Y are defined in claim 1.

FIELD OF THE INVENTION

The invention relates to new serine protease inhibitors, pharmaceuticalcompositions containing the same, as well as to the use of saidinhibitors for the manufacture of a medicament for treating andpreventing thrombin-related diseases.

BACKGROUND OF THE INVENTION

Serine proteases are enzymes which, amongst other things, play animportant role in the blood coagulation cascade. Members of this groupof proteases are for example thrombin, trypsin, factors VIIa, IXa, Xa,XIa, XIIa, and protein C.

Thrombin is the serine protease which regulates the last step in thecoagulation cascade. The prime function of thrombin is the cleavage offibrinogen to generate fibrin monomers, which form an insoluble gel bycross-linking. In addition, thrombin regulates its own production byactivating factors V and VIII earlier in the cascade. It also hasimportant actions at the cellular level, where it acts on specificreceptors to cause platelet aggregation, endothelial cell activation andfibroblast proliferation. Thus thrombin has a central regulatory role inhaemostasis and thrombus formation. Since inhibitors of thrombin mayhave a wide range of therapeutical applications, extensive research hasbeen performed in this area. In the development of synthetic inhibitorsof serine proteases, and more specifically of thrombin, the interest insmall synthetic peptides that are recognized by proteolytic enzymes in amanner similar to that of natural substrates, has increased. As aresult, new peptide-like inhibitors have been prepared, such as thetransition state inhibitors of thrombin.

The search for more effective and more selective thrombin inhibitorscontinues unabated in order to obtain thrombin inhibitors which can beadministered in lower dosages and which have fewer and less severe sideeffects. Furthermore, special attention is paid to oral bioavailability.Potent intravenous thrombin inhibitors are clinically effective in acutecare settings requiring the treatment of thrombin-related diseases.However, particularly the prevention of thrombin-related diseases suchas myocardial infarct, thrombosis and stroke require long-term therapy,preferably by orally dosing an anticoagulant.

Many of the peptide-like serine protease inhibitors, in particularthrombin inhibitors, disclosed in prior publications are based on thesequence -D-Phe-Pro-Arg-, see for example compounds as disclosed byBrady et al. [Bioorganic & Medical Chemistry, 3 (1995), 1063-78] and inU.S. Pat. No. 5,597,804. Thrombin inhibitors may also contain lysineside chains instead of arginine, such as other inhibitors disclosed byBrady et al., and Lewis et al. [Thrombosis and Haemostasis, 74(4)(1995), 1107-12], and further by Jones et al. [J. Enzyme Inhibition, 9(995), 43-60]. In the latter publication it was reported that tripeptidecompounds containing α-keto methyl ester functions are labile compoundsand therefore unfavourable for further development as thrombininhibitors. Also thrombin inhibitors having an aminocyclohexyl moietyinstead of lysine or arginine side chain are known [WO 94/25051]. Fromthese and also other references [e.g. U.S. Pat. No. 5,523,308] a numberof variations at the C-terminus of these peptide-like thrombininhibitors is known. The developments in this field have alreadyimproved the understanding of how to modulate the biological propertiesof this type of thrombin inhibitors. However, although great effort isbeing spend on finding selective thrombin inhibitors having good oralbioavailability there are still few transition state thrombin inhibitorsknown in the art which fulfill these requirements.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that compounds of the formula:

R¹SO₂—B—X—Z—C(O)—Y  (I)

wherein

R¹ is R²OOC—(CHR²)_(m)— or R²NH—CO—(CHR²)_(m)— or is selected from(1-12C)alkyl, (2-12C)alkenyl, which groups may optionally be substitutedwith (3-12C)cycloalkyl, (1-6C)alkoxy, OH, COOR², CF₃ or halogen, andfrom (6-14C)aryl, (7-15C)aralkyl and (8-16)aralkenyl, the aryl groups ofwhich may optionally be substituted with (1-6C)alkyl, (3-8C)cycloalkyl,(1-6C)alkoxy, OH, COOH, CF₃ or halogen;

m is 1, 2 or 3;

each group R² is independently H, (1-12C)alkyl, (3-8C)cycloalkyl,(6-14C)aryl or (7-1 5C)aralkyl, the aryl groups of which may besubstituted with (1-6C)alkyl, (1-6C)alkoxy or halogen;

B is a bond, an amino-acid of the formula —NH—CH[(CH₂)_(p)C(O)OH]—C(O)—or an ester derivative thereof wherein p is 1, 2 or 3, Gly, D-1-Piq,D-3-Piq, D-1-Tiq, D-3-Tiq, D-Atc, Aic, or a L- or D-amino acid having ahydrophobic, basic or neutral side chain;

X is an amino acid with a hydrophobic side chain, glutamine, serine,threonine, a cyclic amino acid optionally containing an additionalheteroatom selected from N, O or S, and optionally substituted with(1-6C)alkyl, (1-6C)alkoxy, benzyloxy or oxo, or X is 2-amino-isobutyricacid, —NR²—CH₂—C(O)— or the fragment

 wherein n is 2, 3, or 4, W is CH or N and R³ is H, (1-6C)alkyl orphenyl which groups may optionally be substituted with hydroxy,(1-6C)alkoxy, COOH, COO(1-6C)alkyl, CONH₂, or halogen;

Z is lysine or 4-aminocyclohexylglycine;

Y is —NH-(1-6C)alkylene-C₆H₅, the phenyl group of which may besubstituted with (1-6C)alkyl, (1-6C)alkoxy or halogen, or Y is —OR⁴ or—NR⁵R⁶, wherein R⁴ is H, (2-6C)alkyl or benzyl, and R⁵ and R⁶ areindependently H, (1-6C)alkoxy, or (1-6C)alkyl optionally substitutedwith halogen, or R⁵ and R⁶ together are (3-6C)alkylene, or R⁵ and R⁶together with the nitrogen atom to which they are bonded are

 wherein V is O, S or SO₂;

or a prodrug thereof or a pharmaceutically acceptable salt thereof, arepotent and selective serine protease inhibitors. Specifically, thecompounds of the present invention are inhibitors of thrombin, of factorVIIa/tissue factor and of factor Xa. Compounds of the invention showimproved pharmacokinetics, and in particular good bioavailability afteroral administration. The α-(2-6C)keto ester compounds which are part ofthe present invention do not show the disadvantages of the previouslyreported labile α-keto methyl ester compounds.

The compounds of the present invention are useful for treating andpreventing thrombin-mediated and thrombin-associated diseases. Thisincludes a number of thrombotic and prothrombotic states in which thecoagulation cascade is activated which include, but are not limited to,deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterialocclusion from thrombosis or embolism, arterial reocclusion during orafter angioplasty or thrombolysis, restenosis following arterial injuryor invasive cardiological procedures, postoperative venous thrombosis orembolism, acute or chronic atherosclerosis, stroke, myocardialinfarction, cancer and metastasis, and neurodegenerative diseases. Thecompounds of the invention may also be used as anticoagulants inextracorporeal blood circuits, as necessary in dialysis and surgery. Thecompounds of the invention may also be used as in vitro anticoagulants.

DETAILED DESCRIPTION OF THE INVENTION

Preferred serine protease inhibitors according to this invention are thecompounds wherein Z is lysine. More preferred are the compounds whereinX is a cyclic amino acid, an amino acid with a hydrophobic side chain,glutamine, serine, threonine, —NR²—CH₂—C(O)—, or the fragment

wherein R³ is H, (1-6C)alkyl or phenyl.

Particularly preferred are the compounds wherein X is proline, leucine,glutamine, threonine, phenylalanine, —NR²—CH₂—C(O)— wherein R² ismethyl, cyclopentyl or cyclohexyl, or the fragment

wherein R³ is H or methyl.

Other preferred compounds are those wherein B is a bond or a D-aminoacid having a hydrophobic or neutral side chain. The most preferredcompounds of the invention are those wherein R¹ is (1-6C)alkyl orbenzyl. Preferably R⁴ in the definition of Y is (2-6C)alkyl or benzyl.In particular preferred are the compounds wherein Y is —OCH(CH₃)₂. Alsopreferred compounds have Y is NH₂.

The term (1-12C)alkyl means a branched or unbranched alkyl group having1 to 12 carbon atoms, such as methyl, ethyl, t-butyl, isopentyl, heptyl,dodecyl, and the like. Preferred alkyl groups are (1-6C)alkyl groups,having 1-6 carbon atoms.

A (2-12C)alkenyl group is a branched or unbranched unsaturatedhydrocarbon group having 2 to 12 carbon atoms. Preferred are(2-6C)alkenyl groups. Examples are ethenyl, propenyl, allyl, and thelike.

The term (1-6C)alkylene means a branched or unbranched alkylene grouphaving 1 to 6 carbon atoms, such as —(CH₂)_(s)— and s is 1 to 6,—CH(CH₃)—, —CH(CH₃)—(CH₂)—, etc. Preferred alkylene groups in thedefinition of Y are ethylene and methylene.

The term (1-6C)alkoxy means an alkoxy group having 1-6 carbon atoms, thealkyl moiety of which has the meaning as previously defined.

The term (3-12C)cycloalkyl means a mono- or bicycloalkyl group having3-12 carbon atoms which cycloalkyl group may optionally be substitutedwith an oxo group. Preferred are (3-8C)cycloalkyl, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclo-octyl, etc.Cyclopentyl and cyclohexyl are even more preferred cycloalkyl groups. Apreferred cycloalkyl substituted alkyl group in the definition of R¹ isthe camphor group.

A (6-14C)aryl group is an aromatic moiety of 6 to 14 carbon atoms. Thearyl group may further contain one or more hetero atoms, such as N, S,or O. Examples of aryl groups are phenyl, naphthyl, (iso)quinolyl,indanyl, and the like.

(7-1 5C)Aralkyl and (8-16C)aralkenyl groups are alkyl and alkenyl groupsrespectively, substituted by one or more aryl groups, the total numberof carbon atoms being 7 to 15 and 8 to 16, respectively.

The term halogen means fluorine, chlorine, bromine or iodine.

The term ester derivative means any appropriate ester derivative,preferably (1-4C)alkyl-esters, such as methyl-, ethyl- ort-butyl-esters.

The terms Atc means 2-aminotetralin-2-carboxylic acid and Aic meansamino indane carboxylic acid. The terms 1- and 3-Tiq mean1,2,3,4-tetrahydroisoquinoline-1- and -3-carboxylic acid, respectively;1- and 3-Piq are perhydroisoquinoline-1- and -3-carboxylic acid,respectively.

The term amino acid having a hydrophobic side chain means an amino acidhaving a side chain being (3-8C)cycloalkyl, (6-14C)aryl or (1-6C)alkyl,which alkyl group may optionally be substituted with one or more(3-8C)cycloalkyl groups or (6-14C)aryl groups. The hydrophobic sidechain may optionally be substituted with one or more substituents, suchas hydroxy, halogen, trifluoromethyl, —OSO₂CF₃, (1-4C)alkyl (forinstance methyl or ethyl), (1-4C)alkoxy (for instance methoxy),phenyloxy, benzyloxy, and the like. Preferred amino acids with ahydrophobic side chain are leucine, valine, cyclohexylalanine,4-methoxy-cyclohexylalanine, cyclo-octylalanine, phenylalanine,D-naphthylalanine, tyrosine, O-methyl tyrosine (or:p-methoxy-phenylalanine), 3,3-diphenylalanine, norleucine and leucine.

Amino acids having a basic side chain are for example, but not limitedto, arginine and lysine, preferably arginine.

The term amino acids having a neutral side chain refers to amino acidssuch as glutamine (Gln), methionine sulfon, asparagine (Asn) and thelike. Preferred are Gln and Asn.

Cyclic amino acids are for example 2-azetidine carboxylic acid, proline,pipecolic acid, 1-amino-1-carboxy-(3-8C)cycloalkane (preferably 4C, 5Cor 6C), 4-piperidine carboxylic acid, 4-thiazolidine carboxylic acid,3,4-dehydro-proline, azaproline, 2-octahydroindole carboxylic acid, andthe like. Preferred are 2-azetidine carboxylic acid, proline, pipecolicacid, 4-thiazolidine carboxylic acid, 3,4-dehydro-proline and2-octahydroindole carboxylic acid. In the definitions, the termsubstituted means: substituted by one or more substituents.

The invention also includes prodrugs of the compounds of formula I,which after administration are metabolized into the active compounds.Suitable prodrugs are for example N-alkoxycarbonyl protected (preferablyN-ethoxycarbonyl) derivatives of the compounds of formula I.

The invention further includes a process for preparing a compound offormula I, comprising coupling of suitably protected amino acids oramino acid analogs, followed by removing the protective groups.

The compounds according to formula I may be prepared in a mannerconventional for such compounds. To that end, suitably Nac protected(and side-chain protected if reactive side-chains are present) aminoacid derivatives or peptides are activated and coupled to suitablycarboxyl protected amino acid or peptide derivatives either in solutionor on a solid support. Protection of the α-amino functions generallytakes place by urethane functions such as the acid-labiletert-butyloxycarbonyl group (Boc), benzyloxycarbonyl (Cbz) group andsubstituted analogs or the base-labile 9-fluorenyl-methyloxycarbonyl(Fmoc) group. The Cbz group can also be removed by catalytichydrogenation. Other suitable amino protective groups include Nps, Bpoc,Msc, etc. A good overview of amino protective groups is given is givenin The Peptides, Analysis, Synthesis, Biology, Vol. 3 E. Gross and J.Meienhofer, Eds., (Academic Press, New York, 1981). Protection ofcarboxyl groups can take place by ester formation e.g. base-labileesters like methyl- or ethylesters, acid labile esters liketert-butylesters, or hydrogenolytically-labile esters like benzylesters.Protection of the side chain function of lysine or4-aminocyclohexylglycine may be accomplished by using the aforementionedgroups. Activation of the carboxyl group of the suitably protected aminoacids or peptides can take place by the azide, mixed anhydride, activeester, or carbodiimide method, especially with the addition of catalyticand racemization-suppressing compounds like 1-hydroxybenzotriazole,N-hydroxysuccinimide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine,N-hydroxy-5-nor-bornene-2,3-dicarboximide. See, e.g. The Peptides,Analysis, Synthesis, Biology (see above) and Pure and Applied Chem.59(3), 331-344 (1987).

The compounds of the invention, which can be in the form of a free base,may be isolated from the reaction mixture in the form of apharmaceutically acceptable salt. The pharmaceutically acceptable saltsmay also be obtained by treating the free base of formula I with anorganic or inorganic acid such as hydrogen chloride, hydrogen bromide,hydrogen iodide, sulfuric acid, phosphoric acid, acetic acid, propionicacid, glycolic acid, maleic acid, malonic acid, methanesulfonic acid,fumaric acid, succinic acid, tartaric acid, citric acid, benzoic acid,and ascorbic acid.

The compounds of this invention possess one or more chiral carbon atoms,and may therefore be obtained as a pure enantiomer, or as a mixture ofenantiomers, or as a mixture containing diastereomers. Methods forobtaining the pure enantiomers are well known in the art, e.g.crystallization of salts which are obtained from optically active acidsand the racemic mixture, or chromatography using chiral columns. Fordiastereomers straight phase or reversed phase columns may be used.

The compounds of the invention may be administered enterally orparenterally, and for humans Preferably in a daily dosage of 0.001-100mg per kg body weight, preferably 0.01-10 mg per kg body weight. Mixedwith pharmaceutically suitable auxiliaries, e.g. as described in thestandard reference, Gennaro et al., Remington's Pharmaceutical Sciences,(18th ed., Mack Publishing company, 1990, see especially Part 8:Pharmaceutical Preparations and Their Manufacture) the compounds may becompressed into solid dosage units, such as pills, tablets, or beprocessed into capsules or suppositories. By means of pharmaceuticallysuitable liquids the compounds can also be applied in the form of asolution, suspension, emulsion, e.g. for use as an injectionpreparation, or as a spray, e.g. for use as a nasal spray.

For making dosage units, e.g. tablets, the use of conventional additivessuch as fillers, colorants, polymeric binders and the like iscontemplated. In general any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused. Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

The invention is further explained by reference to the followingillustrative Examples.

General

Abbreviations

Et=ethyl

Bzl=benzyl

Boc=tert-butyloxycarbonyl

Cbz=benzyloxycarbonyl

Cha=cyclohexylalanyl

Pro=prolyl

Lys=lysyl

Acg=4-aminocyclohexyl glycyl

TFA=trifluoro acetic acid

Pac=phenylacetyl

Nps=nitrophenylsulfonyl

Bpoc=2-p-biphenylisopropyloxycarbonyl

Asp=aspartyl

Glu=glutamyl

Dpa=diphenylalanyl

H-Aad-OH=amino-adipic acid

Tyr(Me)=(O-methyl)-tyrosyl

Phe=phenylalanyl

Nal=naphthylen-2-yl-alaninyl

H-3-Tiq-OH=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

Msc=methylsulfonylethyloxycarbonyl

Teoc=2-(trimethylsilyl)ethoxycarbonyl

norLeu(cyclo)-Gly-OH=(S)-3-amino-2-oxo-hexahydro-1-azepineacetic acid

norVal(cyclo)-Gly-OH=(S)-3-amino-2-oxo-1-piperidineacetic acid

Experimental

The solvent systems used in HPLC are: A: 0.5 M phosphate buffer pH=2.1;B: water; C: acetonitrile/water 3/2 v/v.

Unless stated otherwise the retention times (Rt (LC)) were determined onan analytical HPLC Supelcosil LC-18-DB column (5 μm particles; 250×2.1mm), which was eluted using a gradient (as specified) of solvent systemsA, B and C at a flow rate of 0.25 ml/min at 35° C.

EXAMPLE 1 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-NHBzl (a) Cbz-Lys(Boc)-OMe

To a solution of Cbz-Lys(Boc)-OH (28 g) in dichloromethane/methanol (9/1v/v; 500 mL) was added2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(23.6 g) and the solution was adjusted to pH 8 by addition oftriethylamine. The reaction mixture was stirred for 2 hours at roomtemperature. The mixture was washed successively with cold 1Nhydrochloric acid, water, 5% sodium hydrogencarbonate, and water anddried over sodium sulfate. The filtrate was evaporated and the residuewas chromatographed on silica gel using ethyl acetate/heptane (1/4 v/v)as eluent. The factions containing Cbz-Lys(Boc)-OMe were pooled andevaporated. Yield: 29.1 g

TLC: Rf=0.85, ethyl acetate/heptane=3/1 v/v on silica.

(b) Cbz-Lys (Boc)Ψ[cyanoacetate]

To a cold (−78° C.) solution of Cbz-Lys(Boc)-OMe (29.1 g) in drydichloromethane (800 mL) was added dropwise diisobutylaluminium hydride(222 mL of 1M solution in hexane) keeping the reaction temperature below−70° C. The resulting solution was stirred at −78° C. for 1 hour and anaqueous 5% citric-acid solution (600 mL) was added to the reactionmixture. The two layer mixture was stirred at room temperature for 10minutes, the layers were separated and the aqueous layer was extractedtwice with dichloromethane. The combined dichloromethane layers werewashed with water, dried over sodium sulfate and filtered. The filtratewas stirred under a nitrogen atmosphere and cooled on a icewater-bath. Asolution of sodium cyanide (36.3 g) and benzyltriethyl ammonium chloride(4.2 g) in water (600 mL) was added. Under vigorous stirring aceticanhydride was added portionwise (2×9 mL) over a period of 30 min. Theorganic layer was separated and the aqueous layer was extracted twicewith dichloromethane. The combined dichloromethane layers were washedwith water, dried over sodium sulfate, filtered and evaporated in vacuo.The residue was purified by chromatography on silica (eluentheptane/ethyl acetate=1/1 v/v) to yield Cbz-Lys (Boc)Ψ[cyanoacetate](26.3 g.).

TLC: Rf=0.60, dichloromethane/ethyl acetate=7/3 v/v on silica.

(c) Cbz-Lys(Boc)Ψ[CHOHCO]-OMe

A solution of Cbz-Lys(Boc)Ψ[cyanoacetate] (26.3 g.) indiethylether/methanol=3/1 v/v (600 mL) was cooled to −20° C. under anitrogen atmosphere, and 66 g of gaseous hydrogen chloride wasintroduced keeping the temperature below −5° C. The reaction mixture waskept at 4° C. overnight. Water (100 mL) was added dropwise to thereaction mixture keeping the temperature below 5° C. After stirring for16 h at room temperature the organic layer was separated and washed withwater. The aqueous layer was saturated with sodium chloride andextracted with sec-butanol/dichloromethane=3/2 v/v. The organic phasewas washed with brine, dried over sodium sulfate, filtered andevaporated in vacuo to give 25.4 g of the crude amine. The residue wastaken up in N,N-dimethylformamide (400 mL), di-tert-butyl dicarbonate(16 g) was added and adjusted to pH 8 using triethylamine. The reactionmixture was stirred at room temperature overnight. The solvent wasremoved by evaporation at reduced pressure. The residue was dissolved inethyl acetate, washed with water and brine successively, dried oversodium sulfate, filtered and evaporated in vacuo. The residue waspurified by chromatography on silica (eluent: ethyl acetate/heptane=4/6v/v ) to yield Cbz-Lys(Boc)Ψ[CHOHCO]-OMe (15.8 g).

TLC: Rf=0.75, ethyl acetate/pyridine/acetic acid/water=63/20/6/11v/v/v/v on silica.

(d) Cbz-Lys(Boc)Ψ[CHOHCO]—OH

A stirred solution of Cbz-Lys(Boc)Ψ[CHOHCO]-OMe (2.0 g) indioxane/water=7/3 v/v (50 mL) at room temperature was treatedportionwise with a 2M sodium hydroxide solution (2.36 mL). After 1 hourthe reaction mixture was diluted with water (100 mL), 2M hydrochloricacid was added until pH 2.0 and extracted with dichloromethane. Thecombined organic phases were washed with water, dried over sodiumsulfate, filtered and concentrated in vacuo to yieldCbz-Lys(Boc)Ψ[CHOHCO]—OH (1.85 g).

TLC: Rf=0.65, ethyl acetate/pyridine/acetic acid/water=63/20/6/11v/v/v/v on silica.

(e) Cbz-Lys(Boc)Ψ[CHOHCO]-NHBzl

To a stirred solution of Cbz-Lys(Boc)Ψ[CHOHCO]—OH (0.90 g) inN,N-dimethylformamide (10 mL) were added 1-hydroxybenzotriazole (HOBt,444 mg), N-methylmorpholine (0.5 mL), benzylamine (282 mg) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 462mg). After stirring for 16 hours at room temperature the reactionmixture was poured into water and this aqueous mixture was extractedwith ethyl acetate. The ethyl acetate extract was washed with 1Nhydrochloric acid, water, aqueous 5% sodium hydrogencarbonate and water,dried over sodium sulfate, filtered and concentrated in vacuo to yieldCbz-Lys(Boc)Ψ[CHOHCO]-NHBzl (1.0 g).

TLC: Rf=0.81, ethyl acetate/pyridine/acetic acid/water=163/20/6/11v/v/v/v on silica.

(f) H-Lys(Boc)Ψ[CHOHCO]-NHBzl.HCl

To a solution of Cbz-Lys(Boc)Ψ[CHOHCO]-NHBzl (1.0 g) in methanol (25 mL)were added 10% palladium on activated carbon (100 mg) and 2Mhydrochloric acid (1 mL) and this suspension was hydrogenated atatmospheric pressure for 1 hour at room temperature. The palladiumcatalyst was removed by filtration and the filtrate was concentrated invacuo to yield H-Lys(Boc)Ψ[CHOHCO]-NHBzl.HCl (0.87 g).

TLC: Rf=0.15, ethyl acetate/pyridine/acetic acid/water=163/20/6/11v/v/v/v on silica.

(g) N-Boc-L-α-Amino-ε-caprolactam

To a stirred solution of L-α-Amino-ε-caprolactam (10 g) in dioxane/water(2/1 v/v) (30 mL) was added 1N sodium hydroxide solution (7.8 mL)followed by di-t-butyl dicarbonate (18.8 g). The mixture was stirred for16 hours at room temperature and concentrated in vacuo. The residue wasdissolved in ethyl acetate and washed with water and brine, dried oversodium sulfate, filtered and evaporated in vacuo. The crude material wastriturated by hexane, filtered and dried in vacuo to yieldN-Boc-L-α-Amino-ε-caprolactam (16 g).

TLC: Rf=0.85, ethyl acetate/heptane 1/1 v/v on silica.

(h) Boc-norLeu(cyclo)-Gly-OMe

N-Boc-L-α-Amino-ε-caprolactam (10 g) was dissolved in dichloromethane(100 mL). At −20° C. a 1M solution of lithium bis(trimethylsilyl)amidein tetrahydrofuran/cyclohexane 1/1 v/v (1 equiv.) was added slowly andthe mixture was stirred for 30 min. Methyl bromoacetate (4 mL) wassubsequently added and the mixture was stirred for 2 hours at roomtemperature. Additional lithium bis(trimethylsilyl)amide intetrahydrofuran/cyclohexane 1/1 v/v was added to force the reaction tocompletion. The mixture was diluted by dichloromethane and washed with0.1N hydrochloric acid, water, 5% aqueous sodium hydrogencarbonatesolution and brine, dried over sodium sulfate, filtered and evaporatedin vacuo. The residue was purified by chromatography on silica gel(eluent: heptane/ethyl acetate 6/4 v/v) to yield 12 gBoc-norLeu(cyclo)-Gly-OMe.

TLC: Rf=0.55, ethyl acetate/heptane 6/4 v/v on silica.

(i) BzlSO₂-norLeu(cyclo)-Gly-OMe

Boc-norLeu(cyclo)-Gly-OMe (3 g) was dissolved in TFA/dichloromethane 1/1v/v (30 mL) and stirred for 1 hour at room temperature. The reactionmixture was concentrated in vacuo. The residue was dissolved indichloromethane (25 mL) and a solution of benzylsulfonylchloride (2.25g) in dichloromethane (10 mL) was added slowly at 0° C. Triethylaminewas added to keep the pH at 8 during the reaction. The mixture wasstirred for 1 hour at room temperature, whereafter the mixture wasconcentrated in vacuo. The residue was dissolved in ethyl acetate andwashed with 5% sodium hydrogencarbonate solution, water and brine, driedover sodium sulfate, filtered and evaporated in vacuo. The residue waspurified by chromatography on silica gel (eluent: dichloromethane/ethylacetate 95/5 v/v) to yield BzlSO₂-norLeu(cyclo)-Gly-OMe (3.9 g).

TLC: Rf=0.40, dichloromethane/ethyl acetate 9/1 v/v on silica.

(j) BzlSO₂-norLeu(cyclo)-Gly-OH

A solution of BzlSO₂-norLeu(cyclo)-Gly-OMe (3.9 g) in dioxane/water 9/1(100 mL) at room temperature was treated with sufficient 1N sodiumhydroxide to keep the pH at 13 for 2 hours. After acidification, themixture was poured into water and extracted with dichloromethane. Theorganic layer was washed with water and dried on sodium sulfate Thefiltrate was concentrated to yield 3.6 g of the title compound.

TLC: Rf 0.60, ethyl acetate/pyridine/acetic acid/water 63/20/6/11v/v/v/v on silica.

(k) BzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]-NHBzl

To a cold (0° C.) solution of BzlSO₂-norLeu(cyclo)-Gly-OH (340 mg) inN,N-dimethylformamide (10 mL) were successively added1-hydroxybenzotriazole (HOBt, 203 mg) and dicyclohexylcarbodiimide (DCC,217 mg). After stiring for 30 minutes at 0° C.H-Lys(Boc)Ψ[CHOHCO]-NHBzl.HCl (402 mg), prepared as described under (f),and triethylamine (0.15 mL) were added. The mixture was stirred at 0° C.for 1 hour and then kept at room temperature overnight. The mixture wascooled to −20° C. and dicyclohexylurea was removed by filtration. Thefiltrate was evaporated to dryness. The residue was dissolved in ethylacetate and washed successively with 1M hydrochloric acid, water,aqueous 5% sodium hydrogencarbonate, water and brine, dried over sodiumsulfate and concentrated in vacuo to affordBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]-NHBzl (690 mg).

TLC: Rf=0.75, ethyl acetate/pyridine/acetic acid/water=l163/20/6/11v/v/v/v on silica.

(l) BzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[COCO]-NHBzl

To a solution of BzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]-NHBzl (680mg) in dry dichloromethane (20 mL) was added 424 mg of periodinane(Dess-Martin reagent). After stirring at room temperature for one hour,aqueous 2% sodium thiosulfate solution (20 mL) and aqueous 5% sodiumhydrogencarbonate solution (20 mL) were added and the mixture wasstirred for 30 min at room temperature. The organic layer was separated,washed with water, dried over sodium sulfate, filtered and evaporated invacuo to give crude BzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[COCO]-NHBzl (561mg).

TLC: Rf=0.85, ethyl acetate/pyridine/acetic acid/water=163/20/6/11v/v/v/v on silica.

(m) BzlSO₇-norLeu(cyclo)-Gly-LysΨ[COCO]-NHBzl

BzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[COCO]-NHBzl (560 mg, crude) wastreated with trifluoroacetic acid (10 mL) and stirred for 1 hour at roomtemperature. The reaction mixture was concentrated in vacuo and theresidue dissolved in water and directly charged onto a preparative HPLCDeltaPak RP-C₁₈ column, which was subsequently eluted using a gradientelution system of 20% A/80% B to 20% A/45% B/35% C over 45 min at a flowrate of 80 mL/min. Yield: 287 mg ofBzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]—OH.

Rt (LC): 23.8 min, 20% A/60% B/20%C to 20% A/80% C in 30 min.

EXAMPLE 2 EthylSO₂-D-Cha-Pro-LysΨ[COCO]—OH (a) Boc-D-Cha-Pro-OPac

To a solution of Boc-D-Cha-OH.H₂O (21.5 g) in N,N-dimethylformamide (143mL) at 0° C. were added hydroxybenzotriazole (HOBt) (13.7 g) anddicyclohexylcarbodiimide (DCC) (15.7 g) and stirred at 0° C. for 30minutes. H-Pro-OPac. TFA (20 g) was disolved in 50 mL ofN,N-dimethylformamide, the pH was adjusted to 8 with triethylamine andthis solution was added to the reaction mixture. This was allowed tocontinue for 16 hours during which the temperature was increased to roomtemperature. The mixture was filtered, concentrated in vacuo, dissolvedin ethylacetate, washed with 1N hydrochloric acid, water, 5% sodiumhydrogencarbonate solution and brine, dried over sodium sulfate,filtered and evaporated in vacuo. Yield 28 g.

TLC: Rf=0.5, dichloromethane/methanol 95/5 v/v on silica.

(b) EthylSO₂-D-Cha-Pro-OPac

Boc-D-Cha-Pro-OPac (3.8 g) was dissolved in TFA/dichloromethane 1/1 v/v(25 mL) and stirred for 30 minutes at room temperature. The reactionmixture was evaporated in vacuo. The crude amine was dissolved indichloromethane (50 mL) and ethanesulfonyl chloride (0.8 mL) was addedat −78° C. Triethylamine was added to keep the pH at 8 during thereaction. The mixture was stirred for 3 hours at 0° C., whereafter water(25 mL) was added. After an additional stirring for 30 minutes at roomtemperature, the reaction mixture was concentrated in vacuo. The residuewas dissolved in diethyl ether and washed with 1N hydrochloric acid,water, 5% sodium hydrogencarbonate solution and brine, dried over sodiumsulfate, filtered and evaporated in vacuo. Trituration of the crudematerial with methanol yielded ethylSO₂-D-Cha-Pro-OPac (3.0 g).

TLC: Rf=0.6, dichloromethane/methanol 95/5 v/v on silica.

(c) EthylSO₂-D-Cha-Pro-OH

To a solution of ethylSO₂-D-Cha-Pro-OPac (10 g) in tetrahydrofuran (250mL) was added 1M solution of tetrabutylammonium fluoride intetrahydrofuran (84 mL). The reaction mixture was stirred for 30 minutesat room temperature and poured into water (1 L). The aqueous solutionwas extracted with ethyl acetate. The combined organic layers weresuccessively washed with 1N hydrochloric acid and water, dried oversodium sulfate and concentrated in vacuo. The residue was purified bycrystallisation from ethyl acetate/diisopropylether to yieldEthylSO₂-D-Cha-Pro-OH (6.0 g).

TLC: Rf=0.2, ethyl acetate/pyridine/acetic acid/water 163/20/6/11v/v/v/v on silica.

(d) EthylSO₂-D-Cha-Pro-LysΨ[COCO]—OH

The DCC/HOBt-coupling between EthylSO₂-D-Cha-Pro-OH andH-Lys(Boc)Ψ[CHOHCO]-OMe.HCl, saponification, Dess-Martin oxidation,deprotection and purification were done according to the proceduresdescribed in example 1. Yield: 163 mg of the title compound.

Rt (LC): 36.35 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 3 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-OEt (a) Cbz-Lys(Boc)Ψ[CHOHCO]-OEt

Cbz-Lys(Boc) Ψ[CHOHCO]-OMe (751 mg) was dissolved in 25 mL of 3NHCl/ethanol solution and stirred during 4.5 hours at room temperature.The reaction solution was evaporated to dryness and coevaporated threetimes with ethanol to yield 691 mg of Cbz-Lys Ψ[CHOHCO]-OEt. Thisproduct was dissolved in 10 mL dry dichloromethane and di-tert-butyldicarbonate (425 mg) was added. The pH of the solution was adjusted andmaintained at 8 with triethylamine and the reaction was stirred for 16hours at room temperature. Water was added and the organic layer waswashed and dried to yield 782 mg of the desired product. Afterpurification on silica using heptane/ethyl acetate 2/3 the final yieldwas 696 mg.

TLC: Rf 0.95, ethyl acetate/pyridine/acetic acid/water 232/31/18/7v/v/v/v on silica.

(b) H-Lys(Boc)Ψ[CHOHCO]-OEt.HCl

To a solution of Cbz-Lys(Boc)Ψ[CHOHCO]-OEt (696 mg) in ethanol (25 mL)were added 10% palladium on activated carbon (100 mg) and 2Nhydrochloric acid (0.8 mL) and this suspension was hydrogenated atatmospheric pressure for 50 minutes at room temperature. The palladiumcatalyst was removed by filtration and the filtrate was concentrated invacuo to yield H-Lys(Boc)Ψ[CHOHCO]-OEt.HCl (525 mg).

TLC: Rf=0. 17, ethyl acetate/pyridine/acetic acid/water=232/31/18/7v/v/v/v on silica.

(c) BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-OEt

Coupling with BzlSO₂-norLeu(cyclo)-Gly-OH, oxidation, deprotection andpurification were done according to procedures described in Example 1.Yield: 186 mg of the title compound.

Rt (LC): 32.46 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 4 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]—NH₂

The coupling between BzlSO₂-norLeu(cyclo)-Gly-OH andH-Lys(Boc)Ψ[CHOHCO]—NH₂.HCl. and the subsequent oxidation, deprotectionand purification were done according to procedures described in Example1 to yield 103 mg of the title compound.

Rt (LC): 27.50 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 5 EthylSO₂-D-Cha-Pro-LysTr[COCO]-OEt

The DCC/HOBt-coupling between EthylSO₂-D-Cha-Pro-OH (270 mg) andH-Lys(Boc)Ψ[CHOHCO]-OEt.HCl (268 mg), Dess-Martin oxidation,deprotection using trifluoroacetic acid and purification were doneaccording to the procedures described in Example 1. Yield: 41 mg of thetitle compound.

Rt (LC): 40.7 min. 20% A/80% B to 20% A/20% B/60% C in 40 min. andmaintain this mixture of eluens for an additional 10 min.

EXAMPLE 6 EthylSO₂-D-Cha-Pro-LysΨ[COCO]-NHBzl

The DCC/HOBt-coupling between EthylSO₂-D-Cha-Pro-OH (250 mg) andH-Lys(Boc)Ψ[CHOHCO]-NHBzl.HCl (611 mg), Dess-Martin oxidation,deprotection using trifluoroacetic acid and purification were doneaccording to the procedures described in Example 1. Yield: 208 mg of thetitle compound.

Rt (LC): 28.7 min. 20% A/60% B/20%C to 20% A/80% C in 30 min.

EXAMPLE 7 EthylSO₂-D-Cha-Pro-LysΨ[COCO]—NH₂

The procedures described in Example 1 were used to prepare the titlecompound. H-Lys(Boc)Ψ[CHOHCO]—NH₂.HCl (0.84 g) was prepared fromCbz-Lys(Boc)Ψ[CHOHCO]—OH (0.95 g) as described forH-Lys(Boc)Ψ[CHOHCO]-NHBzl.HCl. Then DCC/HOBt-coupling betweenEthylSO₂-D-Cha-Pro-OH (189 mg) and H-Lys(Boc)Ψ[CHOHCO]—NH₂.HCl (179 mg),Dess-Martin oxidation, deprotection using trifluoroacetic acid andpurification yielded 126 mg of the title compound.

Rt (LC): 36.3 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 8 BzlSO₂-norVal(cyclo)-Gly-LysΨ[COCO]—OH (a)(S)-3-((benzyloxycarbonyl)amino)-2-oxo-piperidine

Cbz-Ornithine-OH.HCl (25 g) was dissolved in 2 L of N,N-dimethylformamide and 12 mL of triethyl amine was added to a pH of 8.5.2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU, 26.5 g) in 250 mL of N,N-dimethyl formamide was added dropwiseunder vigorous stirring. The mixture was allowed to react for 16 hoursat room temperature while continously adjusting the pH with triethylamine to 8.5. The reaction mixture was concentrated to dryness,dissolved in ethyl acetate and washed with 1N hydrochloric acid, water,5% sodium hydrogen carbonate, water and brine, dried on sodium sulfate,filtered and evaporated to dryness to yield 11.7 g of the titlecompound.

TLC: Rf=0.80, ethyl acetate/pyridine/acetic acid/water=63/20/6/11v/v/v/v on silica.

(b) Cbz-norVal(cyclo)-Gly-OMe

(S)-3-((benzyloxycarbonyl)amino)-2-oxo-piperidine (5 g) was dissolved indichloromethane (50 mL). At −20° C. a 1M solution of lithiumbis(trimethylsilyl)amide in tetrahydrofuran/cyclohexane 1/1 v/v (20 mL,1 equiv.) was added slowly and the mixture was stirred for 30 min.Methyl bromoacetate (1.9 mL) was subsequently added and the mixture wasstirred for 30 minutes at room temperature. The mixture was diluted withethyl acetate and quenched with a saturated aquous ammonium chloridesolution. The organic layer was washed with water and brine, dried oversodium sulfate, filtered and evaporated in vacuo. The residue waspurified by chromatography on silica gel (eluent:dichloromethane/methanol 95/5 v/v) to yield 4.7 gCbz-norVal(cyclo)-Gly-OMe.

TLC: Rf=0.38,ethyl acetate/heptane 3/1 v/v on silica.

(c) BzlSO₂-norVal(cyclo)-Gly-OMe

Cbz-norVal(cyclo)-Gly-OMe (4.7 g) was dissolved in 40 mL of methanol,500 mg 10% palladium on charcoal was added, 7.4 mL of a 2N hydrochloricacid was added and hydrogenated at atmospheric pressure for 1 hour atroom temperature. The reaction mixture was filtered, evaporated in vacuoand immediately used in the next step as H-norVal(cyclo)-Gly-OMe.HCl.

The crude amine was dissolved in dichloromethane (50 mL) andbenzylsulfonylchloride (2.82 g) was added slowly at 0° C. Triethylaminewas added to keep the pH at 8 during the reaction. The mixture wasstirred for 1 hour at room temperature, whereafter the mixture waswashed with water and brine, dried over sodium sulfate, filtered andevaporated in vacuo. The residue was purified by chromatography onsilica gel (eluent: dichloromethane/methanol 95/5 v/v) to yieldBzlSO₂-norVal(cyclo)-Gly-OMe (2 g).

TLC: Rf=0.87, ethyl acetate/pyridine/acetic acid/water=63/20/6/11v/v/v/v on silica.

(d) BzlSO₂-norVal(cyclo)-Gly-OH

The saponification of BzlSO₂-norVal(cyclo)-Gly-OMe (2 g) was doneaccording to the procedure described in Example 1. Yield: 1.8 g.

TLC: Rf=0.40, ethyl acetate/pyridine/acetic acid/water=63/20/6/11v/v/v/v on silica.

(e) BzlSO₂-norVal(cyclo)-Gly-LysΨ[COCO]—OH

Coupling between BzlSO₂-norVal(cyclo)-Gly-OH andH-Lys(Boc)Ψ[CHOHCO]-OMe.HCl, saponification, oxidation, deprotection andpurification were done according to procedures described in Example 1.Yield: 107 mg of the title compound.

Rt (LC): 24.45 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 9 EthylSO-D-Cha-Pro-LysΨ[COCO]-O-iPropyl

H-Lys(Boc)Ψ[CHOHCO]—O-i-Propyl.HCl (0.32 g) was prepared using theprocedure described for H-Lys(Boc)Ψ[CHOHCO]-OEt.HCl in Example 3starting from Cbz-Lys(Boc)Ψ[CHOHCO]-OMe (0.49 g) and 2-propanol. TheDCC/HOBt-coupling between EthylSO₂-D-Cha-Pro-OH (239mg) andH-Lys(Boc)Ψ[CHOHCO]—O-i-Propyl.HCl (316 mg), Dess-Martin oxidation,deprotection using trifluoroacetic acid and purification were doneaccording to the procedures described in Example 1. Yield: 123 mg of thetitle compound.

Rt (LC): 43.0 min. 20% A/80% B to 20% A/20% B/60% C in 40 min. andmaintain this mixture of eluens for an additional 10 min.

EXAMPLE 10 BzlSO₂-norVal(cyclo)-Gly-LysΨ[COCO]-Azetidine

The procedures described in Example 1 were used to prepare the titlecompound. Cbz-Lys(Boc)Ψ[CHOHCO]-Azetidine (2.26 g) was prepared fromCbz-Lys(Boc)Ψ[CHOHCO]—OH (2.7 g) as described forCbz-Lys(Boc)Ψ[CHOHCO]-NHBzl. Hydrogenation ofCbz-Lys(Boc)Ψ[CHOHCO]-Azetidine (269 mg) yieldedH-Lys(Boc)Ψ[CHOHCO]-Azetidine.HCl (214 mg). Then DCC/HOBt-couplingbetween BzlSO₂-norVal(cyclo)-Gly-OH (175 mg) andH-Lys(Boc)Ψ[CHOHCO]-Azetidine.HCl (214 mg), Dess-Martin oxidation,deprotection using trifluoroacetic acid and purification yielded 84 mgof the title compound.

Rt (LC): 27.8 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 11 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-Azetidine

Cbz-Lys(Boc)Ψ[CHOHCO]-Azetidine was prepared according to proceduresdescribed in Example 10. The hydrogenation, coupling toBzlSO₂-norLeu(cyclo)-Gly-OH, oxidation, deprotection and purificationwere also done according to procedures described in Example 1. Yield:100 mg of the title compound.

Rt (LC): 33.61 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 12 BzlSO₂-norLeu(cyclo)-Gly-Lys(Ethoxycarbonyl)Ψ[COCO]-Azetidine(a) BzlSO₂-norLeu(cyclo)-Gly-LysΨ[CHOHCO]-Azetidine.TFA

BzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]-Azetidine (prepared accordingto procedures described in Example 10) (220 mg) was dissolved in 10 mLof dichloromethane/trifluoroacetic acid 1/1 v/v and stirred for 2 hoursat room temperature. Solvents were removed by evaporation and theresidue titruated with diethyl ether. Yield: 267 mg.

TLC: Rf=0.57, ethyl acetate/pyridine/acetic acid/water=63/20/6/11v/v/v/v on silica.

(b) Bz1SO₂-norLeu(cyclo)-Gly-Lys(Ethoxycarbonyl)Ψ[CHOHCO]-Azetidine

BzlSO₂-norLeu(cyclo)-Gly-LysΨ[CHOHCO]-Azetidine.TFA (267 mg) wasdissolved in 10 mL of N,N-dimethylformamide and 46 μL ofethylchloroformate was added after which the pH was adjusted to 8.5 withtriethylamine. After stirring for 16 hours at room temperature, thereaction mixture was diluted with ethyl acetate, washed with water, 5%sodium hydrogencarbonate, 2% citric acid and brine, dried on sodiumsulfate, filtered and evaporated to dryness to yield 150 mg of the titlecompound.

TLC: Rf=0.53, dichloromethane/methanol 9/1 v/v on silica.

(c) BzlSO₂-norLeu(cyclo)-Gly-Lys(Ethoxycarbonyl)Ψ[COCO]-Azetidine

Oxidation and purification ofBzlSO₂-norLeu(cyclo)-Gly-Lys(Ethoxycarbonyl)Ψ[CHOHCO]-Azetidine (150 mg)were done according to procedures described in Example 1. Yield 25 mg.

Rt (LC): 26.42 min. 20% A/60% B/20%C to 100% C in 40 min.

EXAMPLE 13 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]—O-iPropyl

Coupling between BzlSO₂-norLeu(cyclo)-Gly-OH (described in Example 1)and H-Lys(Boc)Ψ[CHOHCO]—O-iPropyl.HCl (described in Example 9),oxidation, deprotection and purification were done according toprocedures described in Example 1. Yield: 400 mg of the title compound.

Rt (LC): 40 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 14 BzlSO?-norLeu(cyclo)-Gly-LysΨ[COCO]—NH-iPropyl (a)BzlSO?-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH

The DCC/HOBt-coupling between 1.96 g of BzlSO₂-norLeu(cyclo)-Gly-OH and2.20 g of H-Lys(Boc)Ψ[CHOHCO]-OMe.HCl and saponification of the productwere performed according to the procedures described in example 1.Yield: 3.1 g of the crude title compound.

TLC: Rf=0.4, ethyl acetate/pyridine/acetic acid/water=66/20/6/11 v/v/v/von silica.

(b) BzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—NH-iPropyl

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH and 0.105 mL ofisopropylamine, Dess Martin oxidation (reaction time: 19 h) anddeprotection were done according to the procedures described inexample 1. Yield: 150 mg of the title compound.

Rt(LC): 34.01 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 15 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]—NH-nPropyl

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)TΨ[CHOHCO]—OH and 0.101 mL ofpropylamine, Dess Martin oxidation (reaction time: 24 h) anddeprotection were done according to the procedures described inexample 1. Yield: 144 mg of the title compound.

Rt(LC): 34.22 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 16 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]—NH-Methyl

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH and methylamine (0.4 mL ofa 3 M solution in N,N-dimethylformamide), Dess Martin oxidation(reaction time: 20 h) and deprotection were done according to theprocedures described in example 1. Yield: 127 mg of the title compound.

Rt(LC): 28.36 min. 20% A/80% B to 20% A/20% BI 60% C in 40 min.

EXAMPLE 17 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-pyrrolidinyl

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH and 0.102 mL ofpyrrolidine, Dess Martin oxidation (reaction time: 14 days) anddeprotection were done according to the procedures described inexample 1. Yield: 125 mg of the title compound.

Rt(LC): 36.87 and 37.38 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 18 BzlSO₂-norLeu(cyclo)-Gly-LysyΨ[COCO]-N-Ethyl

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH and ethylamine (1.78 mL ofa 0.7 M solution in N,N-dimethylformamide), Dess Martin oxidation(reaction time: 20 h) and deprotection were done according to theprocedures described in example 1. Yield: 115 mg of the title compound.

Rt(LC): 31.30 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 19 BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-morpholin-4-yl

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH and 0.107 mL ofmorpholine, Dess Martin oxidation (reaction time: 6.5 days) anddeprotection were done according to the procedures described inexample 1. Yield: 148 mg of the title compound.

Rt(LC): 33.73 and 34.17 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 20BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-(1,1-dioxo)thiomorpholin4-yl

To a solution of 2.47 g of thiomorpholine in 25 mL of methanol was added5.75 g of di-tert-butyl dicarbonate and 4 mL of triethylamine. Afterstirring at room temperature for 3h, 50 mL of ethyl acetate was addedand this solution was washed with water adjusted to pH 3 withhydrochloric acid, water, aqueous 5% sodium hydrogencarbonate and brine,dried over magnesium sulfate and concentrated to give 4.73 g ofN-tert-butyloxycarbonyl thiomorpholine. This residue (4.73 g) wasdissolved in 50 mL of dichloromethane and 50 mL of water was added. Tothis stirred mixture was added 11 g 3-chloroperoxybenzoic acid (80-90%purity) in small portions keeping the reaction mixture at pH 7. Afterstirring at room temperature for 16 h the water layer was separated, theorganic layer washed with 5% aqueous sodium thiosulfate, 5% aqueoussodium hydrogencarbonate (three times) and brine, dried over magnesiumsulfate and concentrated. The residue was purified by chromatography onsilica gel (eluent: ethyl acetate/heptanes 2/3 v/v) to give 5.7 g ofN-tert-butyloxycarbonyl thiomorpholine 1,1-dioxide. This sulfon (0.625g) was dissolved in 50 mL of a 3M hydrogenchloride solution in dioxaneand after stirring for 4 hours at room temperature the reaction mixturewas concentrated to give 0.579 g of thiomorpholine 1,1-dioxidehydrochloride.

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH and 0.21 g ofthiomorpholine 1,1-dioxide hydrochloride, Dess Martin oxidation(reaction time: 3 days) and deprotection were done according to theprocedures described in example 1. Yield: 180 mg of the title compound.

Rt(LC): 33.64 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 21

BzlSO₂-norLeu(acico)-Gly-LysΨ[COCO]-N(Methyl)(Methoxy)

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Bc)Ψ[CHOHCO]—OH and 0.12 g ofN,O-dimethylhydroxylamine, Dess Martin oxidation (reaction time: 3.5days) and deprotection were done according to the procedures describedin example 1. Yield: 136 mg of the title compound.

Rt(LC): 33.80 and 34.53 min. 20% A/80% B to 20% A/20% Bt 60% C in 40min.

EXAMPLE 22BzlSO₂-norLeu(cyclo)-Gly-LysΨ[COCO]-(2-(carboxamid)azetidin-1-yl)

The DCC/HOBt-coupling between 1.13 gN-tert-butyloxycarbonyl-L-azetidine-2-carboxylic acid and 1.38 gammmonium chloride was performed as described in example 1 to give 0.468g of N-tert-butyloxycarbonyl-L-azetidine-2-carboxamide. This amide(0.224 g) was dissolved in 5 mL of a 3M hydrogenchloride solution indioxane. After stirring for 3 hours at room temperature the reactionmixture was concentrated to give 0.17 g of azetidine-2-carboxamidehydrochloride.

The EDCI/HOBt-coupling between 0.4 mmolBzlSO₂-norLeu(cyclo)-Gly-Lys(Boc)Ψ[CHOHCO]—OH and 0.17 g ofazetidine-2-carboxamide hydrochloride, Dess Martin oxidation (reactiontime: 20 h) and deprotection were done according to the proceduresdescribed in example 1. Yield: 58 mg of the title compound.

Rt(LC): 26.43 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 23 nPropylSO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl (a)Boc-D-Cha-Pro-OBzl

To a stirred solution of 11.64 g of Boc-D-Cha-OH in 100 mL ofdichloromethane at 0° C. was added 6.36 g of HOBt and 9.72 g of DCC.After 20 minutes a solution of 10.35 g of H-Pro-OBzl.HCl in 40 mL ofdichloromethane adjusted with N,N-diisopropyl ethylamine to pH 8 wasadded. After 16 h the reaction mixture was filtered and the filtrate waswashed successively with water, 0.1 N hydrochloric acid, water, aqueous5% sodium hydrogencarbonate and brine. All aqueous washes were extractedtwice with ethyl acetate, all organic extracts combined, dried oversodium sulfate and concentrated. To the residue was added a mixture ofethyl acetate/heptanes=1/1 (v/v), the resulting suspension filtered andthe filtrate purified by chromatography on silica gel (eluent: ethylacetate/heptanes=1/1 v/v) to yield 19.34 g of Boc-D-Cha-Pro-OBzl.

TLC: Rf=0.8, dichloromethane/methanol=9/1 v/v on silica.

(b) nPropylSO₂-D-Cha-Pro-OBzl

Boc-D-Cha-Pro-OBzl (1.01 g) was dissolved in 42 mL of a 3Mhydrogenchloride solution in dioxane. After stirring for 2 hours at roomtemperature the reaction mixture was concentrated. The residue wasdissolved in 35 mL of dichloromethane and cooled to 0° C. To thisstirred solution was added 0.22 mL of 1-propanesulfonyl chloride and thepH adjusted to 8.5. After stirring for 24 h at room temperature thereaction mixture was concentrated. The residue was dissolved in ethylacetate, washed successively with aqueous 5% sodium hydrogencarbonate,water, aqueous 5% citric acid and brine, dried over magnesium sulfateand concentrated. The crude product was purified by chromatography onsilica gel (eluent: ethyl acetate/heptanes=1/1 v/v) to yield 0.85 g ofnPropylSO₂-D-Cha-Pro-OBzl.

TLC: Rf=0.6, ethyl acetate/heptanes=1/1 v/v on silica.

(c) nPropylSO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

nPropylSO₂-D-Cha-Pro-OBzl (0.85 g) was hydrogenated using the proceduredescribed in example 1 to give 0.54 g of nPropylSO₂-D-Cha-Pro-OH. TheDCC/HOBt coupling of 225 mg of nPropylSO₂-D-Cha-Pro-OH and ofH-Lys(Boc)Ψ[CHOHCO]—O-iPropyl.HCl and Dess Martin oxidation wereperformed according to the procedures described in example 9. TheBoc-group was removed using a 3M hydrogenchloride solution in dioxane asdescribed above and the crude product purified using the preparativeHPLC method described in example 1. Yield: 47 mg of the title compound.

Rt(LC): 27.6 min. 20% A/60% B/20% C to 20% A/80% C in 30 min, then to100% C in 10 min.

EXAMPLE 24 (10-Camphor)SO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

The title compound was prepared from Boc-D-Cha-Pro-OBzl and(−)-10-camphorsulfonyl chloride using the procedures described inexample 23. Yield 12% from Boc-D-Cha-Pro-OBzl.

Rt(LC): 33.6 min. 20% A/60% B/20% C to 20% A/80% C in 30 min, then to100% C in 10 min.

EXAMPLE 25 PhenylSO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

The title compound was prepared from Boc-D-Cha-Pro-OBzl andbenzenesulfonyl chloride using the procedures described in example 23.Yield: 9% from Boc-D-Cha-Pro-OBzl.

Rt(LC): 29.3 min. 20% A/60% B/20% C to 20% A/80% C in 30 min, then to100% C in 10 min.

EXAMPLE 26 Methyl SO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

The title compound was prepared from Boc-D-Cha-Pro-OBzl andmethanesulfonyl chloride using the procedures described in example 23.Yield: 18% from Boc-D-Cha-Pro-OBzl.

Rt(LC): 24.3 min. 20% A/60% B/20% C to 20% A/80% C in 30 min, then to100% C in 10 min.

EXAMPLE 27 iPropyl SO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

The title compound was prepared from Boc-D-Cha-Pro-OBzl andisopropylsulfonyl chloride using the procedures described in example 23.Yield: 2% from Boc-D-Cha-Pro-OBzl.

Rt(LC): 26.8 min. 20% A/60% B/20% C to 20% A/80% C in 30 min, then to100% C in 10 min.

EXAMPLE 28 BenzylSO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

The title compound was prepared from Boc-D-Cha-Pro-OBzl anda-toluenesulfonyl chloride using the procedures described in example 23.Yield: 11% from Boc-D-Cha-Pro-OBzl.

Rt(LC): 30.4 min. 20% A/60% B/20% C to 20% A/80% C in 30 min, then to100% C in 10 mi.

EXAMPLE 29 nButylSO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

The title compound was prepared from Boc-D-Cha-Pro-OBzl and1-butanesulfonyl chloride using the procedures described in example 23.Yield: 29% from Boc-D-Cha-Pro-OBzl.

Rt(LC): 29.3 min. 20% A/60% B/20% C to 20% A/80% C in 30 min, then to100% C in 10 min.

EXAMPLE 30[3-(benzylsulfonylamino)-6-methyl-2-oxo-1,2-dihydropyridinyl]-acetyl-LysΨ[COCO]—O-iPropyl

The DCC/HOBt coupling of 151 mg of[3-(benzylsulfonylamino)-6-methyl-2-oxo-1,2-dihydropyridinyl]-aceticacid (WO 97/01338) and 205 mg of H-Lys(Boc)Ψ[CHOHCO]—O-iPropyl.HCl, DessMartin oxidation, deprotection and purification were performed accordingto the procedures described in example 9 to give 1 mg of the titlecompound.

Rt(LC): 34.7 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 31[3-(benzylsulfonylamino)-2-oxo-1,2-dihydropyridinyl]-acetyl-LysΨ[COCO]—O-iPronyl

The DCC/HOBt coupling of 178 mg of[3-(benzylsulfonylamin6)-2-oxo-1,2-dihydropyridinyl]-acetic acid (WO97/46207) and H-Lys(Boc)Ψ[CHOHCO]—O-iPropyl.HCl and Dess Martinoxidation were performed according to the procedures described inexample 9. Deprotection using hydrogenchloride in dioxane andpurification were performed according to the procedures described inexample 23 to give 116 mg of the title compound.

Rt(LC): 32.4 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 32[3-(benzylsulfonylamino)-6-methyl-2-oxo-1,2-dihydropyridinyl]-acetyl-LysΨ[COCO]—NH₂

The DCC/HOBt coupling of 286 mg of[3-(benzylsulfonylamino)-6-methyl-2-oxo-1,2-dihydropyridinyl]-aceticacid (WO 97/01338) and H-Lys(Boc)Ψ[CHOHCO]-OMe HCl, according to theprocedure described in example 1 yielded 0.51 g of[3-(benzylsulfonylamino)-6-methyl-2-oxo-1,2-dihydropyridinyl]-acetyl-LysΨ[CHOHCO]-OMe.Saponification of this methyl ester, EDCI/HOBt coupling with ammoniumchloride, Dess Martin oxidation, deprotection and purification wereperformed according to the procedures described in example 14 to give 76mg of the title compound.

Rt(LC): 26.9 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 33 BzlSO₂-Aad-Pro-LysΨ[COCO]—OH (a) BzlSO₂-Aad(OtBu)-OH

To a stirred solution of 0.5 g of H-Aad(OtBu)-OH in 4.4 mL of aqueous 1N sodium hydroxide was added 0.42 g of benzylsulfonylchloride in 2 mL ofdioxane. After 16 hours at room temperature, additional 1.4 mL ofaqueous 2 N sodium hydroxide, 0.5 mL of dioxane and 0.09 g ofbenzylsulfonylchloride were added and the reaction mixture stirred foran additional day. The dioxane was removed, water was added, the mixturemade acid (pH 3) using hydrochloric acid and extracted twice withdiethyl ether. The combined ether layers were dried over sodium sulfateand concentrated to give 235 mg of BzlSO₂-Aad(OtBu)-OH.

TLC: Rf=0.7, dichloromethane/methanol/water=14/6/1 v/v/v on silica.

(b) BzlSO₂-Aad(OtBu)-Pro-OH DCC/HOBt coupling of 235 mg ofBzlSO₂-Aad(OtBu)-OH and 168 mg of H-Pro-OBzl.HCl followed byhydrogenation as described in example 23 yielded 193 mg of the titlecompound.

TLC: Rf=0.6, ethyl acetate/pyridine/acetic acid/water#163/20/6/11v/v/v/v on silica.

(c) BzlSO₂-Aad-Pro-LysΨ[COCO]—OH

The DCC/HOBt coupling of 193 mg of BzlSO₂-Aad(OtBu)-Pro-OH andH-Lys(Boc)Ψ[CHOHCO]-OMe HCl, saponification, Dess Martin oxidation,deprotection and purification were performed according to the proceduresdescribed in example 32 to give 85 mg of the title compound.

Rt(LC): 26.1 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 34 BzlSO₂-Glu-Pro-LysΨ[COCO]—OH

Starting with H-Glu(OtBu)-OH according to the route described in example33 gave the title compound. Yield: 3% from H-Glu(OtBu)-OH.

Rt(LC): 22.6 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 35 BzlSO₂-Asp-Pro-LysΨ[COCO]—OH

Starting with H-Asp(OtBu)-OH according to the route described in example33 gave the title compound. Yield: 18% from H-Asp(OtBu)-OH.

Rt(LC): 21.9 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 36 EtSO₂-D-Tyr(Me)-Pro -LysΨ[COCO]—NH (a) EtSO-D-Tyr(Me)-Pro-OH

DCC/HOBt coupling of 2.22 g of Boc-D-Tyr(Me)-OH and 2.0 g ofH-Pro-OBzl.HCl, removal of the Boc protecting group, sulfonylation usingethane sulfonyl chloride and hydrogenation of the benzyl ester using theprocedures described in example 23 yielded 1.0 g of the title compound.

TLC: Rf=0.23, dichloromethane/methanol=95/5 v/v on silica.

(b) EtSO₂-D-Tyr(Me)-Pro -LysΨ[COCO]—NH₂

The DCC/HOBt coupling of 254 mg of EtSO₂-D-Tyr(Me)-Pro-OH andH-Lys(Boc)Ψ[CHOHCO]-OMe.HCl, saponification, EDCI/HOBt coupling withammonium chloride, Dess Martin oxidation, deprotection and purificationwere performed according to the procedures described in example 32 togive 83 mg of the title compound.

Rt(LC): 28.0 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 37 EtSO₂-D-Tyr(Me)-Pro -LysΨ[COCO]—O-iPropyl

The DCC/HOBt coupling of 0.51 g of EtSO₂-D-Tyr(Me)-Pro-OH andH-Lys(Boc)Ψ[CHOHCO]—O-iPropyl.HCl, Dess Martin oxidation, deprotectionand purification were performed according to the procedures described inexample 9 to give 223 mg of the title.

Rt(LC): 36.5 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 38 EtSO₂-D-Tyr(Me)-Pro -LysΨ[COCO]-Azetidine

The DCC/HOBt coupling of 307 mg of EtSO₂-D-Tyr(Me)-Pro-OH andH-Lys(Boc)Ψ[CHOHCO]-Azetidine.HCl, Dess Martin oxidation, deprotectionand purification were performed according to the procedures described inexample 10 to give 83 mg of the title compound.

Rt(LC): 36.4 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 39 EtSO₂-D-Tyr(Me)-Pro -LysΨ[COCO]-N-(4-chloropropyl)

The title compound (43 mg) was obtained as second product in thepurification of example 38.

Rt(LC); 38.1 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 40 BzlSO₂-D-Dpa-Pro-LysΨ[COCO]—O-iPropyl (a) BzlSO₂-D-Dpa-Pro-OH

Removal of the Boc group of 1.5 g of Boc-D-Dpa-Pro-OBzl (WO 97/31937),reaction with benzylsulfonyl chloride and removal of the benzyl esteraccording to the procedures described in example 23 to yield 1.0 g ofthe title compound.

TLC: Rf=0.63, ethyl acetate/pyridine/acetic acid/water=163/20/6/11v/v/v/v on silica.

(b) BzlSO₂-D-Dpa-Pro-LysΨ[COCO]—O-iPropyl

The DCC/HOBt coupling of 0.31 g of BzlSO₂-D-Dpa-Pro-OH andH-Lys(Boc)Ψ[CHOHCO]-O-iPropyl.HCl, Dess Martin oxidation, deprotectionand purification were performed according to the procedures described inexample 9 to give 50 mg of the title compound.

Rt(LC): 32.2 min. 20% A/60% B/20%C to 20% A/80% C in 30 min, then to100% C in 10 min.

EXAMPLE 41 EtSO₂-Leu-Pro-LysΨ[COCO]—O-iPropyl (a) EtSO₂-Leu-OMe

A stirred solution of 3.0 g of H-Leu-OMe.HCl in 30 mL of dichloromethanewas adjusted to pH 8 using triethylamine and cooled at 0° C. Then 3.2 mLof ethanesulfonyl chloride and 2.3 mL of triethylamine were added. Afterstirring for 16 h at room temperature the reaction mixture was washedsuccessively with 0.5 N hydrochloric acid, water and aqueous 5% sodiumhydrogencarbonate and concentrated. The crude product was purified bychromatography on silica gel (eluent: dichloromethane/methanol=9/1 v/v)to yield 3.3 g of EtSO₂-Leu-OMe.

TLC: Rf=0.69, dichloromethane/ethyl acetate=9/1 v/v on silica.

(b) EtSO₂-Leu-Pro-OH

EtSO₂-Leu-OMe (3.3 g) was saponified (procedure example 1), coupled withH-Pro-OBzl (procedure example 23) and the resulting dipetide washydrogenated (procedure example 23) using the indicated procedures togive 3.4 g of the title compound.

TLC: Rf=0.11, dichloromethane/ethyl acetate=9/1 v/v on silica.

(c) EtSO₂-Leu-Pro-LysΨ[COCO]—O-iPropyl

The DCC/HOBt coupling of 145 mg of EtSO₂-Leu-Pro-OH andH-Lys(Boc)Ψ[CHOHCO]—O-iPropyl.HCl, Dess Martin oxidation, deprotectionand purification were performed according to the procedures described inexample 23 to give 120 mg of the title compound.

Rt(LC): 16.6 min. 20% A/60% B/20%C to 20% A/80% C in 30 min.

EXAMPLE 42 BzlSO₂-norLeu(cyclo)-Gly-Acp Ψ[COCO]-Azetidine (a)H-Acg(Boc)[CHOHCO]-OMe.HCl

To a solution of3-[4-(1,1-dimethylethoxycarbonylamino)cyclohexyl]-2-hydroxy-3-nitro-propionicacid methyl ester (Lyle et al, Bioorg. Med. Chem. Lett., 7, 67-72(1997)) (294 mg) in methanol (100 mL) was added 2N hydrochloric acid(0.425 mL) and 10% palladium on activated carbon powder (0.45 g) andthis suspension was hydrogenated at atmospheric pressure at roomtemperature for 16 hours. The palladium catalyst was removed byfiltration and the solvent was removed by evaporation at reducedpressure yielding H-Acg(Boc)Ψ[CHOHCO]-OMe.HCl (289 mg) as a mixture ofdiastereomers.

TLC: Rf=0.26, silica gel, ethyl acetate/pyridine/aceticacid/water=232/31/18/7 v/v/v/v.

(b) BzlSO₂-norLeu(cyclo)-Gly-AcgΨ[COCO]-Azetidine

The DCC/HOBt-coupling between 0.27 g of BzlSO₂-norLeu(cyclo)-Gly-OH and0.25 g of H-Acg(Boc)Ψ[CHOHCO]-OMe.HCl, saponification,EDCI/HOBt-coupling with azetidine hydrochloride, Dess Martin oxidationand deprotection were done according to the procedures described inexample 1. Yield: 82 mg of the title compound.

Rt(LC): 34.8 and 35.4 min. 20% A/80% B to 20% A/20% B/60% C in 40 min.

EXAMPLE 43 EthylSO₂-D-Cha-Pro-AcgΨ[COCO]-OiPropyl (a)Cbz-Acg(aoc)Ψ[CHOHCO]-OMe

A stirred solution of 0.34 g of H-Acg(Boc)Ψ[CHOHCO]-OMe.HCl in 10 mL ofacetonitrile and 10 mL of N,N-dimethylformamide is adjusted to pH 8using N,N-diisopropylethylamine. To this solution 0.24 g ofN-benzyloxycarbonyloxysuccinimide was added. After stirring at roomtemeperature for one hour the reaction mixture was concentrated. Theresidue dissolved in ethyl acetate, washed with water and brine, driedover sodium sulfate and concentrated. The residue was purified bychromatography on silica gel (eluent: ethyl acetate/heptanes 2/3 v/v) togive 0.287 mg of Cbz-Acg(Boc)Ψ[CHOHCO]-OMe.

TLC: Rf=0.25, ethyl acetate/heptanes 1/1 v/v on silica.

(b) Cbz-Acg(Boc)ΨΨ[CHOHCO]-OiPropyl

To a stirred mixture of 5 mL of tetrahydrofuran and 1 mL of 2-propanolunder a nitrogen atmosphere was added slowly added 2.5 mL of a 1.6Nn-butyllithium solution in hexanes. After 20 minutes a solution of 0.28g of Cbz-Acg(Boc)Ψ[CHOHCO]-OMe in 5 mL of 2-propanol was added andstirred for 2 h at room temperature. Then 0.5 mL of acetic acid wasadded and the reaction mixture was concentrated. The residue dissolvedin ethyl acetate, washed with water, dried over sodium sulfate andconcentrated. The residue was purified by chromatography on silica gel(eluent: ethyl acetate/heptanes 2/3 v/v) to give 0.223 mg ofCbz-Acg(Boc)ΨΨ[CHOHCO]-OiPropyl.

TLC: Rf=0.25, ethyl acetate/heptanes 1/2 v/v on silica.

(b) EthylSO₂-D-Cha-Pro-AcgΨ[COCO]-OiPropyl

To a solution of 0.22 g of Cbz-Acg(Boc)Ψ[CHOHCO]-OiPropyl inN,N-dimethylformamide were added 10% palladium on activated carbon (80mg) and 2M hydrochloric acid (0.23 mL) and this suspension washydrogenated at atmospheric pressure for 1 hour at room temperature. Thepalladium catalyst was removed by filtration. This fitrate was used in aDCC/HOBt coupling with 0.166 g of EthylSO₂-D-Cha-Pro-OH using theprocedure described in example 1. The product was oxidised using theDess Martin reagent, the Boc-group removed and purified using theprocedures described in example 1. Yield: 100 mg of the title compound.

Rt(LC): 30.0 min. 20% A/60% B/20% C to 20% A/80% C in 30 min

EXAMPLE 44

Preparation of EtSO₂-B—X-LysΨ[COCO]—O-iPropyl derivatives on solid phase

(a) Teoc-Lys(Boc)Ψ[CHOHCO]—O-iPropyl

Cbz-Lys(Boc)Ψ[CHOHCO]-OMe (10 g) was hydrogenated under the conditionsdescribed in example 1f to afford H-Lys(Boc)Ψ[CHOHCO]-OMe inquantitative yield. The crude product was treated with2-(trimethylsilyl)ethoxycarbonyl hydroxy-succinimide (6.7 g) inN,N-dimethylformamide (100 mL) in the presence ofN,N-diisopropylethylamine (pH=8) for 2 hours at room temperature. Thereaction mixture was evaporated to dryness and the residue was dissolvedin ethyl acetate and washed with 2% aqueous citric acid, water, 5%aqueous sodium hydrogencarbonate and brine. Drying over sodium sulfateand evaporation of the solvent afforded, after chromatography on silicagel (eluent: ethyl acetate/heptane=1/1 v/v), Teoc-Lys(Boc)Ψ[CHOHCO]-OMe(9.1 g). Subsequent transesterification was accomplished by addingdropwise Teoc-Lys(Boc)Ψ[CHOHCO]-OMe (2.8 g) to a stirred mixture ofisopropyl alcohol (5.4 mL), THF (27.1 mL) and 1.6 M n-butyl lithium inhexane (13.6 mL) at room temperature. After 1 hour the reaction mixturewas cooled to 0° C. and glacial acetic acid (2.5 mL) was added. Thereaction mixture was concentrated to a small volume and diluted withethyl acetate, washed with water (2×) and dried over sodium sulfate.Filtration and removal of the solvent in vacuo gave the crude product.Chromatography on silica gel (eluent: ethyl acetate/heptane=1/1 v/v)afforded the title compound (2.9 g).

TLC: Rf=0.53, heptane/ethyl acetate 1/1 v/v on silica.

(b) Teoc-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl

Teoc-Lys(Boc)Ψ[CHOHCO]—O-iPropyl (2.8 g) was dissolved diethyl ether (36mL) and para-toluene sulfonic acid (1.8 g) was added. After 2 hours at30° C. the reaction mixture was evaporated and the residue was dried invacuo to give Teoc-LysΨ[CHOHCO]—O-iPropyl. To a suspension of 4.2 g ofhydroxymethyl-resin (Bachem, 1.02 mmol/g) in 50 mL ofacetonitrile/dichloromethane (1/1 v/v) and triethylamine (1.81 mL) wasadded N,N-disuccinimidyl carbonate (3.36 g). The suspension was shakenfor 2 hours at ambient temperature on an orbital shaker. The resin wasfiltered off and washed with dichloromethane, acetonitrile anddichloromethane (three times each) and dried.Teoc-LysΨ[CHOHCO]—O-iPropyl (see above) was dissolved in 50 mL ofacetonitrile/dichloromethane (1/1 v/v). The pH of the solution wasadjusted to 8 using triethylamine. This solution was added to the resinand the suspension was shaken for 16 hours at room temperature. Thesolvent was removed by filtration and the resin was washed according tothe procedures described earlier. After drying in vacuo, 5.43 g ofTeoc-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl was obtained.

(c) H-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl

A suspension of 2.5 g of Teoc-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropylin trifluoroacetic acid/dichloromethane (50 mL, 1/9 v/v) was shaken for45 min at room temperature. The resin was thoroughly washed withdichloromethane and dried under high vacuum to giveH-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl (2.5 g)

(d) Boc-X-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl

H-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl was divided over 4 reactorsin portions of 500 mg. The resin was washed with a 1% solution ofN,N-diisopropylethylamine in dichloromethane/N,N-dimethylformamide (3/2v/v) and dichloromethane (three times each). Next, 10 mL ofdichloromethane/N,N-dimethylformamide (3/2 v/v) was added to the resinfollowed by building block Boc-X—OH (139 mg Boc-D-leu-OH, 139 mgBoc-Leu-OH, 148 mg Boc-Gln-OH or 159 mg Boc-Phe-OH),2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU, 193 mg) and N,N-diisopropylethylamine (105 μL). The suspensionwas shaken for 90 min at room temperature, whereafter the solvent wasremoved by filtration. The resin was washed withdichloromethane/N,N-dimethylformamide (3/2 v/v), N,N-dimethylformamideand dichloromethane (three times each) and dried.

(e) H—X-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl

The Boc-group of the four different X-blocks was removed under the sameconditions as described for the deprotection of the Teoc-group (seeexample 44c) to give four times 500 mg ofH—X-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl. This resin (500 mg) wasdistributed over 5 reaction vessels.

(f) EtSO₂-B—X-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl

The couplings of the second building block EtSO₂-B—OH (27.0 mgEtSO₂-Asn-OH, 26.8 mg EtSO₂-D-Leu-OH, 30.8 mg EtSO₂-D-Phe-OH, 36.8 mgEtSO₂-Nal-OH and 32.4 mg EtSO₂-D-3-Tiq-OH, prepared according to themethods as described in example 41) were performed under the sameconditions as described in procedure (d), based on 100 mg resin. Afterwork-up, the 20 reaction vessels (resulting from 4 different X blocksand 5 different B blocks) were dried in vacuo.

(g) EtSO₂-B—X-Lys(CO—O-methyl-resin)Ψ[COCO]—O-iPropyl

EtSO₂-B—X-Lys(CO—O-methyl-resin)Ψ[CHOHCO]—O-iPropyl (100 mg) was swollenin a solution of 1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (0.18 M) indimethylsulfoxide (2 mL) and dichloromethane (0.2 mL). The reactionmixture was allowed to shake overnight at room temperature, whereafterthe solvent was removed by filtration. Subsequent washing withdimethylsulfoxide and dichloromethane (three times each) afforded, afterdrying, EtSO₂-B—X-Lys(CO—O-methyl-resin)Ψ[COCO] -O-iPropyl.

(h) EtSO₂-B—X-LysΨ[COCO]—O-iPropyl

A solution of trifluoroacetic acid/thioanisole (2 mL, 10/1 v/v) wasadded to EtSO₂-B—X-Lys(CO—O-methyl-resin)Ψ[COCO]—O-iPropyl (100 mg) andthe reaction mixture was shaken for 4 hours at room temperature. Theresin was filtered, washed with trifluoroacetic acid (three times)whereafter the filtrate was evaporated to dryness in vacuo. The residuewas rinsed with heptane (2 mL) and vigorously stirred whereafter theheptane layer was decanted. This procedure was repeated twice. The crudeproduct was dried and directly applied on a preparative Supelcosil C18DBcolumn (21×250 mm) for purification, using the following conditions:Flow: 20 mL/min; Buffers A: aqueous trifluoroacetic acid 0.1 M, B:water, C: acetonitrile/water 6/4 v/v; Gradient (depending on thepolarity of the product) 3% A—67% B—30% C to 3% A—52% B—45% C in 40 min.UV-detection at 210 nm. The main peaks, corresponding to the desiredcompounds, were isolated and lyophilized to give the purified endproducts as depicted in table 44.

Table 44: Characterization (retention time on reversed phase HPLC andM+H peak in electrospray mass spectrometry) ofEtSO₂-B—X-LysΨ[COCO]—O-iPropyl prepared on Hydroxymethyl-resin. HPLCconditions: Flow: 1.0 mL/min; Buffers A: water, B: acetonitrile/water(6/4 v/v), C: 0.5 M phosphate-buffer pH=2.1, Gradient: 0→45 min 65%A/15% B/20% C→0% A/80% B/20% C. UV-detection at 210 nm.

B Asn D-Leu D-Phe Nal D-3-Tig EtSO₂-B-D- Rt = 17.07 Rt = Rt = 30.89 Rt =38.17 Rt = 33.54 Leu-D/L- min 27.20 min min min min LysΨ M + H = M + H =M + H = M + H = M + H = [COCO] 536.4 535.6 569.4 619.6 581.4 -O-iPropylEtSO₂-B- Rt = 17.17 Rt = Rt = 32.89 Rt = 37.79 Rt = 33.60 Leu-D/L- min30.78 min min min min LysΨ M + H = M + H = M + H = M + H = M + H =[COCO]-O- 536.4 535.6 569.4 619.6 581.4 iPropyl EtSO₂-B- Rt = 5.40 Rt =Rt = 18.05 Rt = 28.44 Rt = 21.27 Gln-D/L- min 15.74 min min min min LysΨM + H = M + H = M + H = M + H = M + H = [COCO] 551.2 550.4 584.4 634.4596.4 -O-iPropyl EtSO₂-B- Rt = 20.75 Rt = Rt = 35.18 Rt = 39.47 Rt =35.92 Phe-D/L- min 33.33 min min min min LysΨ M + H = M + H = M + H =M + H = M + H = [COCO] 570.4 569.4 603.4 653.6 615.6 -O-iPropyl

EXAMPLE 45

The following compounds can be prepared by using the methods of thepresent invention:

CF₃SO₂-D-Cha-Pro-LysΨ[COCO]—O-iPropyl

MeSO₂-D-Tyr(Me)-Pro-LysΨ[COCO]—O-iPropyl

n-ButylSO₂-D-Tyr(Me)-Pro-LysΨ[COCO]—O-iPropyl

CF₃SO₂-D-Tyr(Me)-Pro-LysΨ[COCO]—O-iPropyl

BzlSO₂-D-Tyr(Me)-Pro-LysΨ[COCO]—O-iPropyl

EtSO₂-D-(p-OEt-Phe)-Pro-LysΨ[COCO]—O-iPropyl

EtSO₂-D-Nle-Pro-LysΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Azt-LysΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-(N-cyclopentyl-Gly)-LysΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Val-LysΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Pec-LysΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-(3,4-dehydro-Pro)-LysΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Pro-LysΨ[COCO]-Azetidine

MeSO₂-D-Cha-Pro-LysΨ[COCO]-Azetidine

n-ButylSO₂-D-Cha-Pro-LysΨ[COCO]-Azetidine

CF₃SO₂-D-Cha-Pro-LysΨ[COCO]-Azetidine

BzlSO₂-D-Cha-Pro-LysΨ[COCO]-Azetidine

[3-(BzlSO₂amino)-2-oxo-1,2-dihydropyridinyl]-acetyl-LysΨ[COCO]-Azetidine

[3-(BzlSO₂amino)-6-methyl-2-oxo-1,2-dihydropyridinyl]-acetyl-LysΨ[COCO]-Azetidine

MeSO₂-D-Cha-Pro-AcgΨ[COCO]—O-iPropyl

n-ButylSO₂-D-Cha-Pro-AcgΨ[COCO]—O-iPropyl

CF₃SO₂-D-Cha-Pro-AcgΨ[COCO]—O-iPropyl

BzlSO₂-D-Cha-Pro-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Tyr(Me)-Pro-AcgΨ[COCO]—O-iPropyl

MeSO₂-D-Tyr(Me)-Pro-AcgΨ[COCO]—O-iPropyl

n-ButylSO₂-D-Tyr(Me)-Pro-AcgΨ[COCO]—O-iPropyl

CF₃SO₂-D-Tyr(Me)-Pro-AcgΨ[COCO]—O-iPropyl

BzlSO₂-D-Tyr(Me)-Pro-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Tyr(Et)-Pro-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Nle-Pro-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Azt-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-(N-cyclopentyl-Gly)-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Val-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Pec-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-(3,4-dehydro-Pro)-AcgΨ[COCO]—O-iPropyl

EtSO₂-D-Cha-Pro-AcgΨ[COCO]-Azetidine

EtSO₂-D-Tyr(Me)-Pro-AcgΨ[COCO]-Azetidine

EtSO₂-D-Tyr(Me)-Pro-AcgΨ[COCO]—NH₂

MeSO₂-D-Cha-Pro-AcgΨ[COCO]-Azetidine

n-ButylSO₂-D-Cha-Pro-AcgΨ[COCO]-Azetidine

CF₃SO₂-D-Cha-Pro-AcgΨ[COCO]-Azetidine

BzlSO₂-D-Cha-Pro-AcgΨ[COCO]-Azetidine

3-(BzlSO₂-amino)-1-carboxymethyl-pyridin-2-one-AcgΨ[COCO]—O-iPropyl

3-(BzlSO₂-amino)-1-carboxymethyl-pyridin-2-one-AcgΨ[COCO]-Azetidine

3-(BzlSO₂-amino)-1-carboxymethyl-6-methyl-pyridin-2-one-AcgΨ[COCO]—O-iPropyl

3-(BzlSO₂-amino)-1-carboxymethyl-6-methyl-pyridin-2-one-AcgΨ[COCO]-Azetidine

The biological activities of the compounds of the present invention weredetermined by the following test methods.

I. Anti-thrombin Assay

Thrombin (Factor hIa) is a factor in the coagulation cascade.

The anti-thrombin activity of compounds of the present invention wasassessed by measuring spectrophotometrically the rate of hydrolysis ofthe chromogenic substrate s-2238 exterted by thrombin. This assay foranti-thrombin activity in a buffer system was used to assess theIC₅₀-value of a test compound.

Test medium: Tromethamine-NaCl-polyethylene glycol 6000 (TNP) buffer

Reference compound: 12581 (Kabi)

Vehicle: TNP buffer.

Solubilisation can be assisted with dimethylsulfoxide, methanol,ethanol, acetonitrile or tert.-butyl alcohol which are without adverseeffects in concentrations up to 2.5% in the final reaction mixture.

Technique Reagents*

1. Tromethamine-NaCl (TN) buffer

Composition of the buffer:

Tromethamine (Tris) 6.057 g  (50 mmol) NaCl 5.844 g (100 mmol) Water to1 l

The pH of the solution is adjusted to 7.4 at 37° C. with HCl (10mmol.l⁻¹).

2. TNP buffer

Polyethylene glycol 6000 is dissolved in TN buffer to give aconcentration of 3 g.l^(−1.)

3. S-2238 solution

One vial S-2238 (25 mg; Kabi Diagnostica, Sweden) is dissolved in 20 mlTN buffer to give a concentration of 1.25 mg.ml⁻¹ (2 mmol.l⁻¹).

4. Thrombin solution

Human thrombin (16 000 nKat.vial⁻¹; Centraal Laboratorium voorBloedtransfusie, Amsterdam, The Netherlands) is dissolved in TNP bufferto give a stock solution of 835 nKat.ml⁻¹.

Immediately before use this solution is diluted with TNP buffer to givea concentration of 3.34 nKat.ml⁻¹.

All ingredients used are of analytical grade

For aqueous solutions ultrapure water (Milli-Q quality) is used.

Preparation of Test and Reference Compound Solutions

The test and reference compounds are dissolved in Milli-Q water to givestock concentrations of 10⁻² mol.l⁻¹. Each concentration is stepwisediluted with the vehicle to give concentrations of 10⁻¹, 10⁻⁴ and 10⁻⁵mol.l⁻¹. The dilutions, including the stock solution, are used in theassay (final concentrations in the reaction mixture: 3·10⁻³; 10⁻³;3·10⁻⁴; 10⁻⁴; 3·10⁻⁵; 10⁻⁵, 3·10⁻⁶ and 10⁻⁶ mol.l⁻¹, respectively).

Procedure

At room temperature 0.075 ml and 0.025 ml test compound or referencecompound solutions or vehicle are alternately pipetted into the wells ofa microtiter plate and these solutions are diluted with 0.115 ml and0.0165 ml TNP buffer, respectively. An aliquot of 0.030 ml S-2238solution is added to each well and the plate is pre-heated andpre-incubated with shaking in an incubator (Amersham) for 10 min. at 37°C. Following pre-incubation the hydrolysis of S-2238 is started byaddition of 0.030 ml thrombin solution to each well. The plate isincubated (with shaking for 30 s) at 37° C.. Starting after 1 min ofincubation, the absorbance of each sample at 405 nm is measured every 2min. for a period of 90 min. using a kinetic microtiter plate reader(Twinreader plus, Flow Laboratories).

All data are collected in an IBM personal computer using LOTUS-MEASURE.For each compound concentration (expressed in mol.l⁻¹ reaction mixture)and for the blank the absorbance is plotted versus the reaction time inmin.

Evaluation of responses: For each final concentration the maximumabsorbance was calculated from the assay plot. The IC₅₀-value (finalconcentration, expressed in μmol.l⁻¹, causing 50% inhibition of themaximum absorbance of the blank) was calculated using the logittransformation analysis according to Hafner et al.(Arzneim.-Forsch./Drug Res. 1977, 27(II): 1871-3).

IC₅₀-values of compounds of the present invention are given in thefollowing Table.

Antithrombin Activity

Example IC₅₀ (μmol-1⁻¹) 4 0.09 24 0.01 38 0.11 40 0.02

II. Anti-factor Xa Assay

Activated Factor X (Xa) is a factor in the coagulation cascade. Theanti-Xa activity of compounds of the present invention was assessed bymeasuring spectrophotometrically the rate of hydrolysis of thechromogenic substrate s-2222 exterted by Xa. This assay for anti-Xaactivity in a buffer system was used to assess the IC₅₀-value of thetest compound.

In general the followed procedure and test conditions were analogous tothose of the anti-thrombin assay as described above. Differences areindicated below.

Reference compound: benzamidine

Vehicle: TNP buffer.

Solubilisation can be assisted with dimethylsulfoxide, methanol,ethanol, acetonitrile or tert.-butyl alcohol which are without adverseeffects in concentrations up to 1% (for DMSO) and 2.5% (for the othersolvents) in the final reaction mixture.

Technique Reagents*

3. S-2222 solution

One vial S-2222 (15 mg; Kabi Diagnostica, Sweden) is dissolved in 10 mlwater to give a concentration of 1.5 mg.ml⁻¹ (2 mmol.l⁻¹).

4. Xa solution

Bovine Factor Xa Human (71 nKat.vial⁻¹, Kabi Diagnostica) is dissolvedin 10 ml TNP buffer and then further diluted with 30 ml TNP buffer togive a concentration of 1.77 nKat.ml⁻¹. The dilution has to be freshlyprepared.

Procedure

Instead of the S-2238 solution (in anti-thrombin assay), the aboveS-2222 solution is added to each well in this assay.

Anti-factor Xa Activity

Example IC₅₀ (μmol-1⁻¹) 1 0.64 5 0.28 28 0.02

III. Anti Factor VIIa/Tissue Factor Assay

Vascular damage initiates a series of enzyme generation reactionsultimately leading to the formation of a fibrin gel at the site of theinjury. The primary enzyme generation reaction is the generation ofactivated factor VII (VIIa) from proenzyme factor VII. This activationreaction takes place by an as yet unknown mechanism. One hypothesis isthat small amounts of factor Xa present in plasma, bind to themembrane-bound protein Tissue Factor (TF)—a protein which normally doesnot contact blood but which gets exposed to it by injury—and that thiscomplex of membrane-bound TF and factor Xa activates factor VII (ref.1). The activated Factor VII then also binds to membrane-bound TF andthis intrinsic tenase complex next converts Factor X into Factor Xa.

Thrombosis develops when there is insufficient control of thecoagulation reaction. One way to restore this control is by inhibitingessential coagulation enzymes such as for instance the complex ofmembrane-bound TF and Factor VIIa. Since inhibitors of VIIa or theVIIa/TF complex most likely will also inhibit the tenase complex,inhibitors of the latter complex may also be found by determining theinhibition of VIIa or VIIa/TF by test compounds. A method is describedby which the inhibitory potency of compounds towards VIIa/TF complex canbe established. Test compounds are mixed at various concentrations withfactor VIIa and TF and with a chromogenic substrate, which is known tobe split far better by TF-bound VIIa than by free VIIa. The amidolyticreaction taking place is continuously monitored in a microtiter platereader. Inhibitory potency of the compounds investigated is expressed bythe IC₅₀, defined as the concentration of compounds yielding 50%inhibition of the amidolytic reaction, ninety minutes after the start ofthe reaction.

Reagents

Hepes Buffer

A ten times concentrated Hepes buffer made by dissolving 29.40 gCaCl₂.2H₂O, 47.66 g Hepes, 87.66 g NaCl and 30.00 g polyethyleneglycol(PEG) MW=6000 in 1000 ml aqua bidest. After the solution has been heatedto 37° C., the pH of the buffer is set on 7.40 with help of 10 molarNaOH. The concentrated buffer solution is stored at 4° C. and is stablefor at least two months at this condition. Prior to use the buffer isdiluted in aqua bidest. 1 to 8 to obtain a final concentration in thewells (See test procedure) of 20 mM CaCl₂, 20 mM Hepes, 150 mM NaCl and0.3% PEG6000. If compounds are dissolved and diluted in aqua bidest. oranother vehicle because of an insufficient solubility the Hepes buffercan be diluted 1 to 6 to preserve the same ionic strength in the test.

Recombinant Human Factor VIIa

Recombinant human factor VIIa is obtained from American Diagnostica Inc,Greenwich, Conn. Each vial contains 1.2 mg recombinant human factorVIIa, which is lyophilized from 2 ml buffer composed of 10 mMglycylglycine, 50 mM NaCl, 10 mM CaCl₂, 30 mg/ml mannitol, 0.1% Tween,pH 5.5. The contents of each of these vials is reconstituted with 2 mlaqua bidest. as indicated by the manufacturer. The 2 ml 1.2*10⁻⁵ stocksolution thus obtained is divided in smaller fractions, which are storedat −30° C. At this condition these VIIa samples are stable for at least6 months.

Recombinant Human Tissue Factor

Recombinant human Tissue Factor is obtained from American DiagnosticaInc, Greenwich, Conn. Each vial contains 25 μg recombinant human TissueFactor (non-lipidated, MW 35000 Dalton), which is lyophilized from 1 mlTris/HCl buffer (pH 8.0) composed of 150 mM NaCl, 200 mM mannitol and 10mM CHAPS (Steroid derivative used to solubilize membrane proteins; seeMerck Index). The contents of each vial is reconstituted with 1 ml aquabidest. as indicated by the manufacturer. The 1 ml 7.14×10⁻⁷ M stocksolution thus obtained is divided in smaller fractions, which are storedat −30° C. Thus stored these VIIa samples are stable for at least 67months.

Pefachrome VIIa

Pefachrome VIIa—CH₃SO₂-D-Cha-but-Arg-pNa.AcOH (MW 670.8)—is obtainnedfrom Pentapharm Ltd, Basle, Switzerland, in vials containing 10 μmol ofthis chromogenic substrate. At the day of the experiment the contents ofa vial are dissloved in 8.33 ml aqua bidest., yielding a 1.2 mMolarPefachrome VIIa solution. What remains of this solution is stored at−30° C. and is stable for at least 6 months at this condition.

Recombinant TF/Recombinant VIIa Solution

At the day of the experiment a deep frozen sample of 1.2*10⁻⁵ Mrecombinant VIIa and a deep frozen sample of recombinant human tissuefactor of 7.14*10⁻⁷ is defrosted. The defrosted 7.14*10⁻⁷ solution ofrecombinant human TF is diluted to 4*10⁻⁷ M and 30 μl of this solutionis mixed with 1 μl of the defrosted recombinant VIIa solution of 1.2*1 5and with 449 μl Hepes buffer, yielding a Hepes buffer solutioncontaining 25 nM recombinant VIIa and 25 nM recombinant TF. The amountof 480 μl TF/VIIa solution is sufficient to examine the inhibition ofeight solutions of one test compound. N times this amount is needed toestablish the IC₅₀ of N test compounds.

Preparation of Test Compounds

Test compounds are dissolved in Hepes buffer to give 5*10⁻³ stocksolutions (A). From this solution seven additional solutions withconcentrations of 1.67*10⁻³ M (B), 5.56*10⁻⁴ M (C), 1.85*10⁻⁴ M (D),6.17*10⁻⁵ M (E), 2.06*10⁻⁵ M (F), 6.86*10⁻⁴ M (G) and 2.29*10⁻⁴ M (H)are prepared by diluting each foregoing solution with a factor three inHepes buffer. Such a series of solutions is prepared for the referencecompound Org 34593 and also for each of the N-1 test compounds. Ifconsidered more convenient, other sets of solutions with differentcompound concentrations may be prepared.

Procedure

Compounds are distributed column by column over the microtiter plate andone column of eight wells is reserved for a series of uninhibitedreactions. Hundred μl of Hepes buffer is brought into all (N+1)*8 wellswith an eight channel pipette. Here N is the number of different testcompounds, including the reference compound Org 34593. Hereafter, fiftyμl of the pefachrome VIIa solution of 1.2 mM is added with an eightchannel pipette to the 100 μl Hepes buffer in all of the (N+1)*8 wellsreserved for compound testing and the blank reactions. Then 50 μl ofeach of the eight solutions of the first, second, third up to the N-thcompound is mixed in a descending order of concentrations with thecontents of the first (A) until the eighth well (H) of columns 1, 2, 3,up to N respectively, so as to obtain a one compound per columndistribution with a from top to bottom descending order of compoundconcentrations per column. Finally 50 μl Hepes buffer is added to theeight wells of the N+1 th column reserved for a series of blanks.

After the whole plate has been prepared it is shaken for 1 minute in amicrotiter plate shaker incubator (Amersham) and the solutions arebrought to 37° C. by incubating the plate in the same instrument for 10minutes.

The reactions are initiated by adding 50 μl of the 25 nM VIIa/25 nM TFsolution, which is preheated at 37° C., to each of the (N+1)*8 wellswith help of an eight channel pipette. After the plate is shaken for 30seconds it is placed in a thermostated microtiter plate reader and the405 nm absorbance is read in each well at time intervals of 1 minuteduring 90 minutes. Absorbances are collected in LOTUS 1.2.3, loaded intoa PC connected to the kinetic reader.

Evaluation

The (end-)absorbances measured at 90 minutes are corrected for the blankabsorbances at the beginning of the test by subtraction of thecorresponding first absorbance value measured 1 minute after theinitiation of the reaction. The corrected end absorbances in thepresence (Abs[I]) and absence (Abs[O]) of the test compound areconverted into logit values by calculating +log((Abs[O]/Abs[I])−1) foreach concentration [I] of the test compound. These logit values areplotted against the −log of concentrations of the test compound. Such alogit plot usually displays a linear relationship between 20% and 80%inhibition of the end-absorbance.

The pIC₅₀ value is defined as the −log (concentration in M) od the testcompound for which the logit value is equal to zero. This value iscalculated by linear regression of the logit vs −log [I] relationpreferably around the logit zero value. When the compound tested is soactive towards VIIa/TF that the pIC₅₀ must be calculated byextrapolation instead of interpolation, it is best to prepare anadditional set of dilutions of this test compound and to perform theassay again. This method of calculating a pIC₅₀ value is described byHafner et al. (ref. 2). The corresponding IC₅₀ is calculated as10^(−pI50) and is expressed in Molar.

Quantity Required

About one mg is required to assess the IC₅₀ of a test compound.

Reference Compound

As a reference compound Org 34593 (PPACK) may be used. For this compoundan IC₅₀ of 3*10⁻⁷ M has been established.

References

(1) The structural biology of expression and function of Tissue Factor:Edgington, T. S., et al. in Thrombosis and Haemostasis 66(1), 67-79(1991).

(2) Mathematical analysis of concentration response relationships:Hafner, D. et al. in Arzneim. Forsch./Drug Research 27, 1871-1873(1977).

As a single point measurement of the anti factor VIIa/tissue factoractivity of compounds of the present invention, the percentage ofinhibition at a concentration of 1×10⁻⁵ M is given in the followingTable. For the determination of the percentages, procedures as describedabove were followed.

Anti, factor VIIa/tissue factor activity (percentage inhibition at aconcentration of 1×10⁻⁵ M):

Example percentage inhibition (%) 44) EtSO₂-D-Phe-Leu-LysΨ[COCO]-O- 98iPropyl 44) EtSO₂-Asn-Leu-LysΨ[COCO]-O-iPropyl 56 44)EtSO₂-D-3-Tiq-Phe-LysΨ[COCO]-O- 91 iPropyl 44)EtSO₂-D-Leu-Gln-LysΨ[COCO]-O- 94 iPropyl

What is claimed is:
 1. A compound having the formula IR¹SO₂—B—X—Z—C(O)—Y  (I) wherein R¹ is R²OOC—(CHR²)_(m)— orR²NH—CO—(CHR²)_(m)— or is selected from (1-12C)alkyl, (2-12C)alkenyl,which groups may optionally be substituted with (3-8C)cycloalkyl,(1-6C)alkoxy, OH, COOR², CF₃ or halogen, and from (6-14C)aryl,(7-15C)aralkyl and (8-16C)aralkenyl, the aryl groups of which mayoptionally be substituted with (1-6C)alkyl, (3-8C)cycloalkyl,(1-6C)alkoxy, OH, COOH, CF₃ or halogen; m is 1, 2 or 3; each group R² isindependently H, (1-12C)alkyl, (3-8C)cycloalkyl, (6-14C)aryl or(7-15C)aralkyl, the aryl groups of which may be substituted with(1-6C)alkyl, (1-6C)alkoxy or halogen; B is a bond, an amino-acid of theformula —NH—CH[(CH₂)_(p)C(O)OH]—C(O)— or an ester derivative thereofwherein p is 1, 2 or 3, Gly, D-Atc, Aic, or a L- or D-amino acid havinga hydrophobic, basic or neutral side chain; X is an L-amino acid with ahydrophobic side chain, serine, threonine, a cyclic amino acidoptionally having an additional heteroatom selected from N, O or S, andoptionally substituted with (1-6C)alkyl, (1-6C)alkoxy, benzyloxy or oxo,or X is 2-amino-isobutyric acid, —NR²—CH₂—C(O)— or the fragment

wherein n is 2, 3, or 4, W is CH or N and R³ is H or (1-6C)alkyl; Z islysine or 4-aminocyclohexylglycine; Y is —NH-(1-6C)alkylene-C₆H₅, thephenyl group of which may be substituted with (1-6C)alkyl, (1-6C)alkoxyor halogen, or Y is —OR⁴ or —NR⁵R⁶, wherein R⁴ is H, (2-6C)alkyl orbenzyl, and R⁵ and R⁶ are independently H or (1-6C)alkyl or R⁵ and R⁶together are (3-6C)alkylene; or a prodrug thereof, which afteradministration are metabolized into the active compounds, or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein Z is lysine.
 3. The compound of claim 1, wherein X is a cyclicamino acid, an L-amino acid with a hydrophobic side chain, serine,threonine, —NR²—CH₂—C(O)—, or the fragment

wherein R³ is H, (1-6C)alkyl.
 4. The compound of claim 1, wherein X isproline, leucine, threonine, phenylalanine, —NR²—CH₂—C(O)— wherein R² ismethyl, cyclopentyl or cyclohexyl, or the fragment

wherein R³ is H or methyl.
 5. The compound of claim 1, wherein B is abond, a D-amino acid having a hydrophobic or neutral side chain.
 6. Thecompound of claim 1, wherein R¹ is (1-6C)alkyl or benzyl.
 7. Thecompound of claim 1, wherein Y is —OCH(CH₃)₂.
 8. A pharmaceuticalcomposition, comprising: an effective amount of a compound of claim 1and pharmaceutically suitable auxiliaries.
 9. A process for preparing apharmaceutical composition, comprising: mixing together a compound ofclaim 1 with pharmaceutically acceptable auxiliaries.
 10. A method oftreating thrombin-related diseases in a patient in need thereof,comprising: administering to the patient an effective amount of acompound according to claim 1.